Efficient methods for producing antimicrobial cationic peptides in host cells

ABSTRACT

Endogenously produced cationic antimicrobial peptides are ubiquitous components of host defenses in mammals, birds, amphibia, insects, and plants. Cationic peptides are also effective when administered as therapeutic agents. A practical drawback in cationic peptide therapy, however, is the cost of producing the agents. The methods described herein provide a means to efficiently produce cationic peptides from recombinant host cells. These recombinantly-produced cationic peptides can be rapidly purified from host cell proteins using anion exchange chromatography.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of allowed U.S. patent applicationSer. No. 09/444,281, filed Nov. 19, 1999, which application claimspriority from U.S. Provisional Application Ser. No. 60/109,218, filedNov. 20, 1998; and each of these applications is incorporated byreference in its entirety.

TECHNICAL FIELD

The present invention relates generally to methods for obtainingrecombinant peptides and proteins from host cells. In particular, thepresent invention relates to improved processes for producing andpurifying cationic peptides from recombinant host cells in which thepeptide is expressed in high yield and is easily recovered.

BACKGROUND OF THE INVENTION

Antimicrobial peptides, particularly cationic peptides have receivedincreasing attention as a new pharmaceutical substance, because of theirbroad spectrum of antimicrobial activities and the rapid development ofmulti-drug-resistant pathogenic microorganisms. Endogenous peptideantibiotics are ubiquitous components of host defenses in mammals,birds, amphibia, insects, and plants. These endogenous antimicrobialpeptides are usually cationic amphipathic molecules that contain 10 to45 amino acid residues and an excess of lysine and arginine residues.(for a review, see Broekaert et al., Plant Physiol. 108:1353, 1995; Ganzand Lehrer, Pharmacol. Ther. 66:191, 1995; Martin et al., J. Leukoc.Biol. 58:128, 1995; Hancock and Lehrer, TIBTECH 16:82, 1998). Examplesof cationic peptides include rabbit defensin, crab tachyplesin, bovinebactenecin, silk-moth cecropin A, frog magainins, and bovineindolicidin. The main site of action of the peptides is the cytoplasmicmembrane of bacteria and other microbes. Due to their amphipathicnature, the peptides disrupt the membrane, causing a loss of potassiumions, membrane depolarization, and a decrease in cytoplasmic ATP.

Since their de novo synthesis or release from storage sites can beinduced rapidly, cationic peptides are particularly important in theinitial phases of resistance to microbial invasion. Cationic peptidesare also effective when administered as therapeutic agents. In thetreatment of topical infection, for example, an α-helical magaininvariant peptide has been shown to be effective against polymicrobic footulcer infections in diabetics, and a protegrin-derived peptide was founduseful for treatment of oral polymicrobic ulcers in cancer patients(Hancock and Lehrer, TIBTECH 16:82, 1998). Efficacy against systemicinfection has been shown with an α-helical peptide used to treatPseudomonas aeruginosa peritoneal infection, a β-sheet protegrin againstmethicillin-resistant Staphylococcus aureus and againstvancomycin-resistant Enterococcus faecalis, and extended-helixindolicidin against Aspergillus fungal infections (Gough et al., InfectImmun. 64:4922, 1996; Steinberg et al., Antimicrob. Agents Chemother.41:1738, 1997; and Ahmad et al., Biochim. Biophys. Acta 1237:109, 1995).Therefore, naturally-occurring cationic peptides, and their syntheticvariants, are valuable antimicrobial therapeutics.

A practical drawback in cationic peptide therapy is the lack of a costeffective, mass-production method of the agents. Typically, theisolation of cationic peptides from natural sources is notcost-effective, and does not apply to the production of engineeredcationic peptide variants which may have increased efficacy. Whilechemical peptide synthesis can be used to manufacture either natural orengineered cationic peptides, this approach is very costly.

Therefore, alternate, more economical and efficient methods of synthesisare needed, such as in vivo synthesis in host cells using recombinantDNA methods. Researchers have attempted various methods for recombinantproduction of cationic peptides. For example, cationic peptides havebeen produced in bacteria, such as E. coli or Staphylococcus aureus,yeast, insect cells, and transgenic mammals (Piers et al., Gene 134:7,1993, Reichhart et al., Invertebrate Reprod. Develop. 21:15, 1992,Hellers et al., Eur. J. Biochem. 199:435, 1991, and Sharma et al., Proc.Nat'l Acad. Sci. USA 91:9337, 1994).

Much attention has focused on production in E. coli, since those versedin the art are familiar with the fact that high productivity can beobtained in E. coli using the recombinant DNA technology. However, forsmall peptides it is often necessary to produce them as part of a largerfusion protein. In this technique the gene for the peptide is joined tothat of a larger carrier protein and the fusion expressed as a singlelarger protein. Following synthesis the peptide must be cleaved from thefusion partner. There is an extensive body of literature on proteinfusion, especially in the gene expression host E. coli. For example, anumber of recombinant proteins have been produced as fusion proteins inE. coli, such as, insulin A and B chain, calcitonin, Beta-globin,myoglobin, and a human growth hormone (Uhlen and Moks, “Gene Fusions forPurposes of Expression, An Introduction” in Methods in Enzymology185:129-143 Academic Press, Inc. 1990). Nevertheless, recombinant geneexpression from a host cell presents a number of technical problems,particularly if it is desired to produce large quantities of aparticular protein. For example, if the protein is a cationic peptide,such peptides are very susceptible to proteolytic degradation, possiblydue to their small size or lack of highly ordered tertiary structure.One approach to solving this problem is to produce recombinant cationicproteins in protease-deficient E. coli host cell strains (see, forexample, Williams et al., U.S. Pat. No. 5,589,364, and WO 96/04373). Yetthere is no general way to predict which protease-deficient strains willbe effective for a particular recombinant protein.

In principle the recombinant DNA technique is straight forward. However,any sequence that interferes with bacterial growth through replicationor production of products toxic to the bacteria, such as lytic cationicpeptides, are problematic for cloning. Foreign peptide gene productsthat are unstable or toxic, like cationic peptides, can also bestabilized by expressing the peptides as part of a fusion proteincomprising a host cell protein. For example, Callaway et al. et al.,Antimicrob. Agents Chemother. 37:1614, 1993, expressed cecropin A in E.coli as a fusion peptide with a truncated portion of the L-ribulokinasegene product, Piers et alet al., Gene 134:7, 1993, expressed fusionproteins in E. coli that comprised glutathione-S-transferase and eitherdefensin (HNP-1) or a synthetic cecropin-melittin hybrid, while Hara etal., Biochem. Biophys. Res. Commun. 220:664, 1996, expressed silkwormmoricin in E. coli as a fusion protein with a α-galactosidase or amaltose-binding protein moiety.

One of the better options to avoid the toxic effects of a bacteriolyticpeptide on the host bacterial cells in highly efficient production, andto avoid proteolytic degradation of the peptides, is to utilize theintrinsic bacterial host mechanism of driving heterologous proteins intoinclusion bodies as a denatured insoluble form.

The approach outlined above suffers from the inherent limitation onoverall productivity imposed by the use of a small single peptide (circa10%) in the large fusion protein.

Accordingly, a need exists for a means to efficiently produce cationicpeptides from recombinant host cells.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compositions and methods for expressinglarge quantities of a selected polypeptide. Within one aspect of theinvention, large quantities of a selected polypeptide can be expressedutilizing a multi-domain fusion protein expression cassette whichcomprises a promoter operably linked to a nucleic acid molecule which isexpressed as an insoluble protein, wherein the nucleic acid moleculeencodes a polypeptide comprising the structure (cationicpeptide)-[(cleavage site)-(cationic peptide)]_(n), wherein n is aninteger having a value between 1 and 100. Within certain embodiments, acleavage site may be inserted on either side of the structure, e.g.,(cleavage site)-(cationic peptide)-[(cleavage site)-(cationicpeptide)]_(n) cleavage site, wherein n is an integer having a valuebetween 1 and 100.

Within certain embodiments, utilizing the methods described herein, theunit: -(cleavage site)-(cationic peptide)- can be added to the aboveexpression cassette in order to specifically add a defined number ofcationic sequences to be expressed. Within various embodiments, n is aninteger having a value of 2, 3, 5, 10, or, 20 on the lower end, and 10,15, 20, 30, 40, 50, 75, or 80 on the upper end (e.g., n may be aninteger between about 2 and 30, 2 and 40, etc., 5 and 30, 6, or, 7 and40, etc., up to 10 or 20 to 40, 50, 70 or 80). As an example, within oneembodiment n has a value of between 5 and 40 or 10 and 40.

Within certain embodiments, the nucleic acid molecule may furthercomprise a carrier protein. Within various embodiments, to the extentthat a carrier protein is to be expressed by the expression cassette, itcan be located at either the N-terminus or the C-terminus of the fusionprotein. A wide range of carrier proteins can be utilized, including forexample, a cellulose binding domain (CBD), or, a fragment of CBD. Withinvarious embodiments, the carrier protein can be greater than, equal to,or less than 100 amino acids in length.

Within further embodiments, the cleavage sites within the expressioncassette can be cleaved by, for example, low pH, or, by a reagent suchas cyanogen bromide,2-(2-nitrophenylsulphenyl)-3-methyl-3′-bromoindolenine, hydroxylamine,o-iodosobenzoic acid, Factor Xa, thrombin, enterokinase, collagenase,Staphylococcus aureus V8 protease, endoproteinase Arg-C, or trypsin.

Within another embodiment, the expression cassette may more specificallybe comprised of (a) a carrier protein, (b) an anionic spacer peptidecomponent having at least one peptide with the structure (cleavagesite)-(anionic spacer peptide), and (c) a cationic peptide componenthaving at least peptide with the structure (cleavage site)-(cationicpeptide) wherein the cleavage site can be on either side of the anionicspacer peptide or cationic peptide, and elements (a), (b), and (c) canbe in any order and or number. Within a further related embodiment, theexpression cassette may be comprised of (a) an anionic spacer peptidecomponent having at least one peptide with the structure (cleavagesite)-(anionic spacer peptide), and (b) a cationic peptide componenthaving at least peptide with the structure (cleavage site)-(cationicpeptide), wherein the cumulative charge of said anionic spacer peptidecomponent reduces the cumulative charge of said cationic peptidecomponent.

To the extent an anionic spacer is included, such a spacer may have, 0,1, 2, or more cysteine residues. Within certain embodiments, there canbe more, the same number, or fewer anionic spacers than cationicpeptides in the fusion construct. Within certain embodiments, theanionic spacer is smaller in size than the cationic peptide.

A wide variety of cleavage sites can be utilized, including for example,a methionine residue. In addition, a wide variety of promoters can beutilized, including for example the lacP promoter, tacP promoter, trcPpromoter, srpP promoter, SP6 promoter, T7 promoter, araP promoter, trpPpromoter, and λ promoter.

The present invention also provides methods for producing fusionproteins utilizing the above-described expression cassettes. Within oneembodiment, such methods generally comprise the step of culturing arecombinant host cell containing an expression cassette, underconditions and for a time sufficient to produce the fusion protein.Representative examples of suitable host cells include yeast, fungi,bacteria (e.g., E. coli), insect, and plant cells.

Once the fusion protein has been produced, it may be further purifiedand isolated. Further, the fusion protein may be cleaved into itsrespective components (e.g., utilizing low pH, or, a reagent such ascyanogen bromide,2-(2-nitrophenylsulphenyl)-3-methyl-3′-bromoindolenine, hydroxylamine,o-iodosobenzoic acid, Factor Xa, thrombin, enterokinase, collagenase,Staphylococcus aureus V8 protease, endoproteinase Arg-C, or trypsin).

Further, the fusion protein or cleaved cationic peptide may be purifiedutilizing a chromatographic method (e.g., an anion chromatography columnor resin). Within certain embodiments, the column can be charged with abase, and washed with water prior to loading the column with saidcationic peptide. Within various embodiments, the column can beequilibrated with water and up to about 8 M urea. Moreover, the cationicpeptide is solubilized in a solution comprising up to about 8 M urea.Within further embodiments, the cationic peptide is solubilized in asolution comprising a mild organic solvent, such as, for example,acetonitrile, or, an alcohol such as methanol or ethanol.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are identified below and areincorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the maps of: (A) plasmid pET21(+), (B) plasmid pET-CBD180, (C)a PCR fragment containing cbd180 and (D) plasmids pET21CBD180-B andpET21 CBD180-X.

FIG. 2 shows maps of fusion poly cationic peptide genes.

FIG. 3 is an SDS-PAGE analysis showing the expression of differentCBD-poly-MBI-11 fusion proteins. Column ST: molecular weight markers:14.4, 21.5, 31, 45, 66.2 and 97.4 kDa; Column 1: whole cell lysate of E.coli MC4100 (pGP1-2) cultivated at 30° C.; Column 2: whole cell lysateof E. coli MC4100 (pGP1-2, pET21CBD96-11) cultivated at 30° C.; Column3: whole cell lysate of induced E. coli MC4100 (pGP1-2, pET21CBD96-11)at 42° C.; Column 4: whole cell lysate of E. coli MC4100 (pGP1-2, pET21CBD96-2x11) cultivated at 30° C.; Column 5: whole cell lysate of inducedE. coli MC4100 (pGP1-2, pET21CBD96-2x11) at 42° C.; Column 6: whole celllysate of E. coli MC4100 (pGP1-2) cultivated at 30° C.; Column 7: wholecell lysate of E. coli MC4100 (pGP1-2, pET21CBD96-3×11) cultivated at30° C.; Column 8: whole cell lysate of induced E. coli MC4100 (pGP1-2,pET21CBD96-3×11) at 42° C.; Column 9: whole cell lysate of E. coliMC4100 (pGP1-2, pET21CBD96-4x11) cultivated at 30° C.; Column 10: wholecell lysate of induced E. coli MC4100 (pGP1-2, pET21 CBD96-4x11) at 42°C.;

FIG. 4 shows maps of cassettes used for construction of genes ofmulti-domain proteins.

FIG. 5 presents maps of plasmid pET21CBD96 and one of its inserts.

FIG. 6 shows maps of fusion multi-domain protein genes.

FIG. 7 is SDS-PAGE analyses showing the results of fermentation ofmulti-domain clones having five or more MBI-11B7 copies. The upperpanels represent the multidomain clones fused to CBD carrier. The lowerpanels show the multi-domain clones carrier-free. The left panels showthe whole cell lysates, where the right side panels show the inclusionbodies partitioning step. The major band in each lane represents therelevant multidomain protein and the “x” numbers appearing at the bottomof each lane indicate the number of the MBI— peptide copies. Numbersappearing along the left edge of the gels represents molecular weightstandards (kD).

FIG. 8 shows maps of portions of plasmids pET21-3s-5x11B7 andpET21-5s-7x11B7.

FIG. 9 is a chromatogram of the Q-Sepharose chromatography step forcationic peptide purification, which monitors UV absorbance at 280 nmand conductivity.

FIG. 10 is a schematic drawing that illustrates the construction ofplasmids pET21CBD-X and pET21CBD-B.

FIG. 11 is a graph showing the results of reverse-phase analysis of theQ-Sepharose chromatography leading peak, representing pure cationicpeptide. In this study, a C8-column (4.6×10, Nova-Pak, Waters) wasequilibrated with 0.1% TFA in water at 1 ml/min flow rate. Then 50 μl ofQ-Sepharose chromatography leading peak material, diluted with 50 μlequilibration solution, was loaded on the column. Elution was performedwith a 0-45% gradient of solution B (0.1% TFA, 99.9% Acetonitrile) at 1%increase B per min, then step to 100% B.

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

As discussed above, a successful approach to stabilizing foreign peptidegene products which are inherently unstable or toxic is to express themfused to a protein which displays stability in the relevant host cell.In the case of small cationic peptides, however, production of a fusionprotein will lead to a small portion of the desired peptide and anapparent low yield. A major gain in productivity and therefore economicsof the process can be made if the fraction of desired peptide in thefusion protein is substantially greater. A favored route for thisconcept concerns expression of a fusion protein containing multiplesequential copies (a concatomer or multi-domain protein) of the peptideseparated by linker sequences. The linkers are the points at which theconcatomer (multi-domain protein) will be cleaved to give monomers ofthe desired peptide with most probably modified C-termini as a result ofthe cleavage process.

On the other hand, increasing the number of copies of a cationic peptideper fusion protein will make it a more and more basic protein, which mayeffect the expression of the fusion protein and/or increase its toxicityfor the host cell.

An approach to overcome the high basicity of the recombinantly-producedmulti-domain cationic protein, and also decrease its toxicity, is toinclude small acidic peptide sequences in the linker sequences thatneutralize the positive charge of the cationic peptide. To keep theeconomic concept of high ratio of the cationic peptide in themulti-domain protein it is important to engineer the acidic peptide tobe as small as possible, preferably smaller than the cationic peptide.The natural phenomenon of a multipeptide precursor structure consistingof cationic peptide and anionic spacer has been described(Casteels-Josson et al (1993) EMBO J., vol. 12, 1569-1578). In thispublication the authors describe the natural production of apidaecin, anantibacterial cationic peptide, in insects such as the honeybee (Apismellifera). Apidaecin is generated as a single gene comprising multiplerepeated precursor units, each consisting of an apidaecin peptide gene(18 amino acids) preceded by an acidic spacer region (6-8 amino acids).In a further example, Lee et al., Protein Exp. Purif 12:53 (1998),expressed in E. coli six copies of the cationic peptide buforin II perfusion protein, which also included as acidic peptide modified magaininintervening sequences that alternated with the cationic peptidesequences. The magainin intervening sequences were “modified” in thatthe sequences included flanking cysteine residues. According to Lee etal., the “presence of cysteine residues in the acidic peptide wascritical for the high level expression of the fusion peptide multimers.”

In initial studies, the present inventors used carrier proteins ofdifferent sizes to express monomer and polymer forms of cationicpeptides. The test carrier protein of these studies were CBD and afragment of the same derived from Clostridium cellulovorans cellulosebinding protein A. The chosen carrier protein fulfilled the requirementsof high expression and accumulation in E. coli as insoluble forms. Thisapproach was limited by a significant decrease in expression when thenumber of cationic fused peptide genes exceeded three copies. There wasessentially no expression from vectors containing more than four copiesof a peptide gene. A new procedure was designed which allowed themultiplication of relevant cationic peptide genes using a specificanionic spacer sequence that encoded a negatively charged peptide. Inthese studies, the anionic spacer peptide consisted of 11 amino acids.Various genes encoding cationic peptide-anionic spacer peptidemulti-domain proteins were constructed and fused to the carrier protein.A high level of expression was achieved for all constructs harboringmore than thirty copies of the relevant cationic peptide gene. Insubsequent studies, polymers of cationic peptide genes with anionicspacers were liberated from the carrier and expressed directly. Theseconstructs achieved high levels of expression and a high percentage oftarget cationic peptide in the carrier-free multi-domain protein.

2. Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

A “structural gene” is a nucleotide sequence that is transcribed intomessenger RNA (mRNA), which is then translated into a sequence of aminoacids characteristic of a specific polypeptide.

As used herein, “nucleic acid” or “nucleic acid molecule” refers to anyof deoxyribonucleic acid (DNA), ribonucleic acid (RNA),oligonucleotides, fragments generated by the polymerase chain reaction(PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acids can becomposed of monomers that are naturally-occurring nucleotides (such asdeoxyribonucleotides and ribonucleotides), or analogs ofnaturally-occurring nucleotides (e.g., α-enantiomeric forms ofnaturally-occurring nucleotides), or a combination of both. An “isolatednucleic acid molecule” is a nucleic acid molecule that is not integratedin the genomic DNA of an organism. For example, a DNA molecule thatencodes a cationic peptide that has been separated from the genomic DNAof a cell is an isolated DNA molecule. Another example of an isolatednucleic acid molecule is a chemically-synthesized nucleic acid moleculethat is not integrated in the genome of an organism.

An “isolated polypeptide or protein” is a polypeptide that isessentially free from contaminating cellular components, such ascarbohydrate, lipid, nucleic acid (DNA or RNA) or other proteinaceousimpurities associated with the polypeptide in nature. Preferably theisolated polypeptide is sufficiently pure for clinical injection at thedesired dose. Whether a particular cationic polypeptide preparationcontains an isolated cationic polypeptide can be determined utilizingmethods such as Urea/acetic acid polyacrylamide gel electrophoresis andCoomassie Brilliant Blue staining of the gel, reverse phase highpressure liquid chromatography, capillary electrophoresis, nucleic aciddetection assays, and the Limulus Amebocyte Lysate test. Utilizing sucha method an isolated polypeptide preparation will be at least about 95%pure polypeptide.

An “insoluble polypeptide” refers to a polypeptide that, when cells arebroken open and cellular debris precipitated by centrifugation (e.g.,10,000 g to 15,000 g), produces substantially no soluble component, asdetermined by SDS polyacrylamide gel with Coomassie Blue staining.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ region ofa gene, proximal to the transcriptional start site of a structural gene.If a promoter is an inducible promoter, then the rate of transcriptionincreases in response to an inducing agent. In contrast, the rate oftranscription is not regulated by an inducing agent if the promoter is aconstitutive promoter.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, which has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites at whichforeign nucleotide sequences can be inserted in a determinable fashionwithout loss of an essential biological function of the vector, as wellas nucleotide sequences encoding a marker gene that is suitable for usein the identification and selection of cells transformed with thecloning vector. Marker genes typically include genes that provideantibiotic resistance.

An “expression vector” is a nucleic acid molecule (plasmid, cosmid, orbacteriophage) encoding a gene that is expressed in a host cell.Typically, gene expression is placed under the control of a promoter,and optionally, under the control of at least one regulatory element.Such a gene is said to be “operably linked to” the promoter. Similarly,a regulatory element and a promoter are operably linked if theregulatory element modulates the activity of the promoter.

A “recombinant host” may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells that have beengenetically engineered to contain the cloned gene(s) in the chromosomeor genome of the host cell.

As used herein, “cationic peptide” refers to a peptide that possesses anisoelectric point (pI) of 9 and above. A cationic peptide is at leastfive amino acids in length, and has at least one basic amino acid (e.g.,arginine, lysine, histidine). Cationic peptides commonly do not havemore than 50 amino acids, and typically contain 10 to 35 amino acidresidues.

A “carrier protein” is an amino acid sequence that can be individuallyexpressed in host cells and, by recombinant fusion to a desired peptideor polypeptide can act as a carrier, enabling the expression of thedesired peptide in host cells.

An “anionic spacer peptide domain” is a peptide sequence that issufficiently anionic to decrease the positive charge of an associatedcationic peptide. That is, the combination of a cationic peptide and ananionic spacer peptide has a net charge that is essentially slightlypositive, negative or neutral. The size of an anionic spacer domain issimilar to but preferably smaller than the size of the cationic peptidedomain.

As used herein, a “fusion protein” is a hybrid protein expressed by anucleic acid molecule comprising nucleotide sequences of at least twogenes. A “multi-domain protein” comprises a combination of preferablymore than one “cationic peptide domain,” and an equal, smaller or highernumber of “anionic spacer peptide domains” with suitable cleavage sitesfor separating cationic peptide from the rest of the multi-domainprotein. The multi-domain protein can be fused to a carrier protein toachieve higher expression and/or stability. If stability and expressionlevel of multi-domain protein are satisfactory, there is no need to usea carrier protein. An “anionic spacer peptide component” comprises atleast one anionic spacer peptide with a cleavage site. The “cumulativecharge” of a cationic peptide component refers to the total charge ofall cationic peptides that comprise the cationic peptide component.Similarly, the “cumulative charge” of an anionic spacer peptidecomponent refers to the total charge of all anionic spacer peptides thatcomprise the anionic spacer peptide component.

As used herein, “antimicrobial activity” refers to the ability to killor to prevent the growth of a microbe, or to kill or to prevent thegrowth of microbe-infected cells. The term “microbe” includes bacteria,fungi, yeast, algae, protozoa, and viruses. This term includes but willnot be limited to all these interpretive descriptions of the biologicalactivity of the cationic peptide.

3. Construction and Expression of Vectors Comprising Cationic PeptideGenes

a. Cationic Peptide Expression Vectors

The present invention contemplates the production of “cationic peptide,”as that term is defined above. For example, suitable cationic peptidesinclude but are not limited to, naturally occurring cationic peptidesand analogs thereof, cecropins, normally made by lepidoptera(Steiner etal., Nature 292:246, 1981) and diptera (Merrifield et al., Ciba Found.Symp. 186:5, 1994), by porcine intestine (Lee et al., Proc. Nat'l Acad.Sci. USA 86:9159, 1989), by blood cells of a marine protochordate (Zhaoet al., FEBS Lett. 412:144, 1997), synthetic analogs of cecropin A,melittin, and cecropin-melittin chimeric peptides (Wade et al., Int. J.Pept. Protein Res. 40:429, 1992), cecropin B analogs (Jaynes et al.,Plant Sci. 89:43, 1993), chimeric cecropin A/B hybrids (Düring, Mol.Breed. 2:297, 1996), magainins (Zasloff, Proc. Nat'l Acad. Sci USA84:5449, 1987), cathelin-associated antimicrobial peptides fromleukocytes of humans, cattle, pigs, mice, rabbits, and sheep (Zanetti etal., FEBS Lett. 374:1, 1995), vertebrate defensins, such as humanneutrophil defensins [HNP 1-4], paneth cell defensins of mouse and humansmall intestine (Oulette and Selsted, FASEB J. 10:1280, 1996; Porter etal., Infect. Immun. 65:2396, 1997), vertebrate β-defensins, such asHBD-1 of human epithelial cells (Zhao et al., FEBS Left. 368:331, 1995),HBD-2 of inflamed human skin (Harder et al., Nature 387:861, 1997),bovine β-defensins (Russell et al., Infect. Immun. 64:1565, 1996), plantdefensins, such as Rs-AFP1 of radish seeds (Fehlbaum et al., J. Biol.Chem. 269:33159, 1994), α- and β-thionins (Stuart et al., Cereal Chem.19:288, 1942; Bohlmann and Apel, Annu. Rev. Physiol. Plant Mol. Biol.42:227, 1991), γ-thionins (Broekaert et al., Plant Physiol. 108:1353,1995), the anti-fungal drosomycin (Fehlbaum et al., J. Biol. Chem.269:33159, 1994), apidaecins, produced by honey bee, bumble bee, cicadakiller, hornet, yellow jacket, and wasp (Casteels et al., J. Biol. Chem.269:26107, 1994; Levashina et al., Eur. J. Biochem. 233:694, 1995),cathelicidins, such as indolicidin from bovine neutrophils (Falla etal., J. Biol. Chem. 277:19298, 1996), bacteriocins, such as nisin(Delves-Broughton et al., Antonie van Leeuwenhoek J. Microbiol. 69:193,1996), and the protegrins and tachyplesins, which have antifungal,antibacterial and antiviral activities (Tamamura et al., Biochim.Biophys. Acta 1163:209, 1993; Aumelas et al., Eur. J. Biochem. 237:575,1996; Iwanga et al., Ciba Found. Symp. 186:160, 1994). Illustrativecationic peptides are listed in Table 1. TABLE 1 ILLUSTRATIVE CATIONICPEPTIDES** SEQ Group Name Peptide Sequence ID Reference* AbaecinsAbaecin YVPLPNVPQPGRRPFPTFP 37 Casteels et al. (1990) +TL, 52GQGPFNPKIKWPQGY Andropins Andropin VFIDILDKVENAIHNAAQVGI 38 Samakovliset al. (1991) GFAKPFEKLINPK Apidaecins Apidaecin IA GNNRPVYIPQPRPPHPRI39 Casteels et al. (1989) Apidaecin IB GNNRPVYIPQPRPPHPRL 40 Casteels etal. (1989) Apidaecin II GNNRPIYIPQPRPPHPRL 41 Casteels et al. (1989) ASAS-48 7.4 kDa Galvez et al. (1989) Bactenecins Bactenecin RLCRIWIRVCR 42Romeo et al. (1988) Bac Bac5 RFRPPIRRPPIRPPFYPPFRP 43 Frank et al.(1990) PIRPPIFPPIRPPFRPPLRFP Bac7 RRIRPRPPRLPRPRPRPLPF 44 Frank et al.(1990) PRPGPRPIPRPLPFPRPGPR PIPRPLPFPRPGPRPIPRP BactericidinsBactericidin B2 WNPFKELERAGQRVRDAVI 45 Dickinson et al. (1988)SAAPAVATVGQAAAIARG* Bactericidin B-3 WNPFKELERAGQRVRDAIIS 46 Dickinsonet al. (1988) AGPAVATVGQAAAIARG Bactericidin B-4 WNPFKELERAGQRVRDAIIS 47Dickinson et al. (1988) AAPAVATVGQAAAIARG* Bactericidin B-WNPFKELERAGQRVRDAVI 48 Dickinson et al. (1988) 5P SAAAVATVGQAAAIARGG*Bacteriocins Bacteriocin 4.8 kDa Takada et al. (1984) C3603 Bacteriocin5 kDa Nakamura et al. (1983) IY52 Bombinins BombininGIGALSAKGALKGLAKGLAZ 49 Csordas & Michl (1970) HFAN* BLP-1GIGASILSAGKSALKGLAKGL 50 Gibson et al. (1991) AEHFAN* BLP-2GIGSAILSAGKSALKGLAKGL 51 Gibson et al. (1991) AEHFAN* BombolitinsBombolitin BIIKITTMLAKLGKVLAHV* 52 Argiolas & Pisano (1985) BombolitinBII SKITDILAKLGKVLAHV* 53 Argiolas & Pisano (1985) BPTI BovineRPDFCLEPPYTGPCKARIIR 54 Creighton and Charles PancreaticYFYNAKAGLCQTFVYGGCR (1987) Trypsin Inhibitor AKRNNFKSAEDCMRTCGGA (BPTI)Brevinins Brevinin-IE FLPLLAGLAANFLPKIFCKIT 55 Simmaco et al. (1993) RKCBrevinin-2E GIMDTLKNLAKTAGKGALQS 56 Simmaco et al. (1993) LLNKASCKLSGQCCecropins Cecropin A KWKLFKKIEKVGQNIRDGIIK 57 Gudmundsson et al.AGPAVAWGQATQIAK* (1991) Cecropin B KWKVFKKIEKMGRNIRNGIV 58 Xanthopouloset al. (1988) KAGPAIAVLGEAKAL* Cecropin C GWLKKLGKRIERIGQHTRDA 59Tryselius et al. (1992) TIQGLGIAQQAANVAATARG * Cecropin DWNPFKELEKVGQRVRDAVI 60 Hultmark et al. (1982) SAGPAVATVAQATALAK*Cecropin P₁ SWLSKTAKKLENSAKKRISE 61 Lee et al. (1989) GIAIAIQGGPRCharybdtoxins Charybdtoxin ZFTNVSCTTSKECWSVCQR 62 Schweitz et al. (1989)LHNTSRGKCMNKKCRCYS Coleoptericins Coleoptericin 8.1 kDa Bulet et al.(1991) Crabrolins Crabrolin FLPLILRKIVTAL* 63 Argiolas & Pisano (1984)α-Defensins Cryptdin ILRDLVCYCRSRGCKGRERM 64 Selsted et al. (1992)NGTCRKGHLLYTLCCR Cryptdin 2 LRDLVCYCRTRGCKRRERM 65 Selsted et al. (1992)NGTCRKGHLMYTLCCR MCP1 WCACRRALCLPRERRAGF 66 Selsted et al. (1983)CRIRGRIHPLCCRR MCP2 WCACRRALCLPLERRAGF 67 Ganz et al. (1989)CRIRGRIHPLCCRR GNCP-1 RRCICTTRTCRFPYRRLGTC 68 Yamashita & Saito (1989)IFQNRVYTFCC GNCP-2 RRCICTTRTCRFPYRRLGTC 69 Yamashita & Saito (1989)LFQNRVYTFCC HNP-1 ACYCRIPACIAGERRYGTCIY 70 Lehrer et al. (1991)QGRLWAFCC HNP-2 CYCRIPACIAGERRYGTCIY 71 Lehrer et al. (1991) QGRLWAFCCNP-1 WCACRRALCLPRERRAGF 72 Ganz et al. (1989) CRIRGRIHPLCCRR NP-2WCACRRALCLPLERRAGF 73 Ganz et al. (1989) CRIRGRIHPLCCRR RatNP-1VTCYCRRTRCGFRERLSGA 74 Eisenhauer et al. (1989) CGYRGRIYRLCCR RatNP-2VTCYCRSTRCGFRERLSGA 75 Eisenhauer et al. (1989) CGYRGRIYRLCCRβ-Defensins BNBD-1 DFASCHTNGGICLPNRCPG 76 Selsted et at. (1993)HMIQIGICFRPRVKCCRSW BNBD-2 VRNHVTCRINRGFCVPIRCP 77 Seisted et al. (1993)GRTRQIGTCFGPRIKCCRSW TAP NPVSCVRNKGICVPIRCPGS 78 Diamond et al. (1991)MKQIGTCVGRAVKCCRKK Defensins- Sapecin ATCDLLSGTGINHSACAAHC 79 Hanzawa etal. (1990) insect LLRGNRGGYCNGKAVCVCRN Insect defensinGFGCPLDQMQCHRHCQTIT 80 Bulet et al. (1992) GRSGGYCSGPLKLTCTCYRDefensins- Scorpion GFGCPLNQGACHRHCRSIR 81 Cociancich et al. (1993)scorpion defensin RRGGYCAGFFKQTCTCYRN Dermaseptins DermaseptinALWKTMLKKLGTMALHAGK 82 Mor et al. (1991) AALGAADTISQTQ DiptericinsDiptericin 9 kDa Reichhardt et al. (1989) Drosocins DrosocinGKPRPYSPRPTSHPRPIRV 83 Bulet et al. (1993) Esculentins EsculentinGIFSKLGRKKIKNLLISGLKN 84 Simmaco et al. (1993) VGKEVGMDWRTGIDIAGCK IKGECIndolicidins Indolicidin ILPWKWPWWPWRR* 85 Selsted et al. (1992)Lactoferricins Lactoferricin BFKCRRWQWRMKKLGAPSIT 86 Bellamy et al.(1992b) CVRRAF Lantibiotics Nisin ITSISLCTPGCKTGALMGCN 87 Hurst (1981)MKTATCHCSIHVSK Pep 5 TAGPAIRASVKQCQKTLKAT 88 Keletta et al. (1989)RLFTVSCKGKNGCK Subtilin MSKFDDFDLDWKVSKQDS 89 Banerjee & Hansen (1988)KITPQWKSESLCTPGCVTG ALQTCFLQTLTCNCKISK Leukocins Leukocin A-valKYYGNGVHCTKSGCSVNW 90 Hastings et al. (1991) 187 GEAFSAGVHRLANGGNGFWMagainins Magainin I GIGKFLHSAGKFGKAFVGEI 91 Zasloff (1987) MKS*Magainin II GIGKFLHSAKKFGKAFVGEI 92 Zasloff (1987) MNS* PGLaGMASKAGAIAGKIAKVALKAL* 93 Kuchler et al. (1989) PGQ GVLSNVIGYLKKLGTGALNA94 Moore et al. (1989) VLKQ XPF GWASKIGQTLGKIAKVGLKE 95 Sures and Crippa(1984) LIQPK Mastoparans Mastoparan INLKALAALAKKIL* 96 Bernheimer & Rudy(1986) Melittins Melittin GIGAVLKVLTTGLPALISWIK 97 Tosteson & TostesonRKRQQ (1984) Phormicins Phormicin A ATCDLLSGTGINHSACAAHC 98 Lambert etal. (1989) LLRGNRGGYCNGKGVCVCRN Phormicin B ATCDLLSGTGINHSACAAHC 99 Lambert et al. (1989) LLRGNRGGYCNRKGVCVRN Polyphemusins Polyphemusin IRRWCFRVCYRGFCYRKCR* 100 Miyata et al. (1989) Polyphemusin IIRRWCFRVCYKGFCYRKCR* 101 Miyata et al. (1989) Protegrins Protegrin IRGGRLCYCRRRFCVCVGR 102 Kokryakov et al. (1993) Protegrin IIRGGRLCYCRRRFCICV 103 Kokryakov et al. (1993) Protegrin IIIRGGGLCYCRRRFCVCVGR 104 Kokryakov et al. (1993) Royalisins RoyalismVTCDLLSFKGQVNDSACAA 105 Fujiwara et al. (1990) NCLSLGKAGGHCEKGVCICRKTSFKDLWDKYF Sarcotoxins SarcotoxinIA GWLKKIGKKIERVGQHTRD 106 Okada &Natori (1985b) ATIQGLGIAQQAANVAATAR* Sarcotoxin IB GWLKKIGKKIERVGQHTRD107 Okada & Natori (1985b) ATIQVIGVAQQAANVAATAR* Seminal SeminalpiasminSDEKASPDKHHRFSLSRYA 108 Reddy & Bhargava (1979) plasminsKLANRLANPKLLETFLSKWI GDRGNRSV Tachyplesins TachyplesinIKWCFRVCYRGICYRRCR* 109 Nakamura et al. (1988) Tachyplesin IIRWCFRVCYRGICYRKCR* 110 Muta et al. (1990) Thionins Thionin BTH6KSCCKDTLARNCYNTCRFA 111 Bohlmann et al. (1988) GGSRPVCAGACRCKIISGPKCPSDYPK Toxins Toxin 1 GGKPDLRPCIIPPCHYIPRPK 112 Schmidt et al. (1992)PR Toxin 2 VKDGYIVDDVNCTYFCGRN 113 Bontems etal. (1991)AYCNEECTKLKGESGYCQW ASPYGNACYCKLPDHVRTK GPGRCH*Argiolas and Pisano, JBC 259:10106 (1984); Argiolas and Pisano, JBC260:1437 (1985); Banerjee and Hansen, JBC 263:9508 (1988); Bellamy etal., J. Appl. Bacter. 73:472 (1992); Bernheimer and Rudy, BBA 864:123(1986); Bohlmann et al., EMBO J. 7:1559 (1988); Bontems et al., Science254:1521 (1991); Bulet et al., JBC 266:24520 (1991); Bulet et al., Eur.J. Biochem. 209:977 (1992); Bulet et al., JBC 268:14893 (1993); Casteelset al., EMBO J. 8:2387 (1989); Casteels et al.,#Eur. J. Biochem. 187:381 (1987); Csordas and Michl, Monatsh Chemisty101:82 (1970); Diamond et al., PNAS 88:3952 (1991); Dickinson et al.,JBC 263:19424 (1988); Eisenhauer et al., Infect, and Imm. 57:2021(1989); Frank et al., JBC 26518871 (1990); Fujiwara et al., JBC265:11333 (1990); Galvez et al., Antimicrobial Agents and Chemotherapy33:437 (1989); Ganz et al.,J.Immunol. 143:1358 (1989); Gibson et al.,JBC 266:23103 (1991); Gudmundsson et al., JBC 266:11510 (1991); #Hanzawaet al., FEBS Letter Bacteriology 173:7491 (1991); Hultmark et al., Eur.J. Biochem. 127:207 (1982); Hurst, Adv. Appl. Micro. 27:85 (1981);Kaletta et al., Archives of Microbiology 152:16 (1989); Kokryakov etal., FEBS Letters 327:231 (1993); Kuchler et al., Fur. J. Biochem.179:281 (1989); Lambert et al., PNAS 86:262 (1989); Lee et al., PNAS86:9159 (1989); Lehrer et al., Cell 64:229 (1991); Miyata et al., J.Biochem. 106:663 (1989); Moore et al., JBC 266:19851 #(1991); Mor etal., Biochemisty 30:108:261 (1990); Nakamura et al., JBC 263:16709(1988); Nakamura et al., Infection and Immunity 39:609 (1983); Okada andNatori, Biochem. J. 229:453 (1985); Reddy and Bhargava, Nature 279:725(1979); Reichhart et al., Eur. J. Biochem. 182:423 (1989); Romeo et al.,JBC 263:9573 (1988); Samakovlis et al., EMBO J. 10:163 (1991); Schmidtet al., Toxicon 30:1027 (1992); Schweitz et al., Biochem. 28:9708(1989); Selsted et al., JBC 258:14485 #(1983); Selsted et al., JBC267:429 324:159 (1993); Sures and Crippa, PNAS 81:380 (1984); Takada etal., Infect. and Imm. 44:370 (1984); Tosteson and Tosteson, BiophysicalJ. 45:112 (1984); Tryselius et al., Eur. J. Biochem. 204:395 (1992);Xanthopoulos et al., Eur. J. Biochem. 172:371 (1988); Yamashita andSaito, lnfect. and 1mm. 57:2405 (1989); Zasloff, PNAS 84:5449 (1987).**See also U.S Patent Nos. 4,822,608; 4,962,277; 4,980,163; 5,028,530;5,096,886; 5,166,321; 5,179,078; 5,202,420; 5,212,073; 5,242,902;5,254,537; 5,278,287; 5,300,629; 5,304,540; 5,324,716; 5,344,765;5,422,424; 5,424,395; 5,446,127; 5,459,235; 5,464,823; 5,466,671;5,512,269; 5,516,682; 5,519,115; 5,519,116; 5,547,939; 5,556,782;5,610,139; 5,645,966; 5,567,681; 5,585,353; 5,589,568; 5,594,103,5,610,139; 5,631,144; 5,635,479; 5,656,456; 5,707,855; 5,731,149;#5,714,467; 5,726,155; 5,747,449; Publication Nos. WO 89/00199; WO90/11766; WO 90/11771; WO 91/00869; WO 91/12815; W~ 91/17760; WO94/05251; WtJ 94/05156; WcJ 94/07528; WO 95/21601; WO 97/00694; WO97/11713; WO 97/18826; WO 97/02287; WcJ 98/03192; WO 98/07833; WO98/07745; WO 98/06425 European Application Nos. EP 17785; 349451;607080; 665239; and Japanese Patent/Patent Application Nos. 4341179;435883; 7196408; 798381; and 8143596.

Nucleic acid molecules encoding cationic peptides can be isolated fromnatural sources or can also be obtained by automated synthesis ofnucleic acid molecules or by using the polymerase chain reaction (PCR)with oligonucleotide primers having nucleotide sequences that are basedupon known nucleotide sequences of cationic peptides. In the latterapproach, a cationic peptide gene is synthesized using mutually priminglong oligonucleotides (see, for example, Ausubel et al., (eds.), ShortProtocols in Molecular Biology, 3 Edition, pages 8-8 to 8-9 (John Wiley& Sons 1995), “Ausubel (1995)”). Established techniques using thepolymerase chain reaction provide the ability to synthesize DNAmolecules of at least two kilobases in length (Adang et al., PlantMolec. Biol. 21:1131, 1993; Bambot et al., PCR Methods and Applications2:266, 1993; Dillon et al., “Use of the Polymerase Chain Reaction forthe Rapid Construction of Synthetic Genes,” in Methods in MolecularBiology, Vol. 15: PCR Protocols: Current Methods and Applications, White(ed.), pages 263-268, (Humana Press, Inc. 1993); Holowachuk et al., PCRMethods Appl. 4:299, 1995).

As noted above, analogs of natural cationic peptides can also berecombinantly produced by the presently described methods. The presenceof a codon may have an adverse effect on expression and therefore a DNAsequence encoding the desired cationic peptide is optimized for aparticular host system, in this case E. coli. Amino acid sequences ofnovel cationic peptides are disclosed, for example, by Falla et al., WO97/08199, and by Fraser et al., WO 98/07745.

One type of cationic peptide analog is a peptide that has one or moreconservative amino acid substitutions, compared with the amino acidsequence of a naturally occurring cationic peptide. For example, acationic peptide analog can be devised that contains one or more aminoacid substitutions of a known cationic peptide sequence, in which analkyl amino acid is substituted for an alkyl amino acid in the naturalamino acid sequence, an aromatic amino acid is substituted for anaromatic amino acid in the natural amino acid sequence, asulfur-containing amino acid is substituted for a sulfur-containingamino acid in the natural amino acid sequence, a hydroxy-containingamino acid is substituted for a hydroxy-containing amino acid in thenatural amino acid sequence, an acidic amino acid is substituted for anacidic amino acid in the natural amino acid sequence, a basic amino acidis substituted for a basic amino acid in the natural amino acidsequence, or a dibasic monocarboxylic amino acid is substituted for adibasic monocarboxylic amino acid in the natural amino acid sequence.

Among the common amino acids, for example, a “conservative amino acidsubstitution” is illustrated by a substitution among amino acids withineach of the following groups: (1) glycine, alanine, valine, leucine, andisoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine andthreonine, (4) aspartate and glutamate, (5) glutamine and asparagine,and (6) lysine, arginine and histidine.

Nucleotide sequences encoding such “conservative amino acid” analogs canbe obtained, for example, by oligonucleotide-directed mutagenesis,linker-scanning mutagenesis, mutagenesis using the polymerase chainreaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22; andMcPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL Press1991)). The antimicrobial activity of such analogs can be determinedusing a standard method, such as the assays described herein.Alternatively, a cationic peptide analog can be identified by theability to specifically bind anti-cationic peptide antibodies.Typically, cationic peptide analogs should exhibit at least 50%, andpreferably, greater than 70, 80 or 90%, of the activity of thecorresponding naturally occurring cationic peptide.

Although one objective in constructing a cationic peptide variant may beto improve its activity, it may also be desirable to alter the aminoacid sequence of a naturally occurring cationic peptide to enhance itsproduction in a recombinant host cell. For example, a nucleotidesequence encoding a radish cationic peptide may include a codon that iscommonly found in radish, but is rare for E. coli. The presence of arare codon may have an adverse effect on protein levels when the radishcationic peptide is expressed in recombinant E. coli. Methods foraltering nucleotide sequences to alleviate the codon usage problem arewell known to those of skill in the art (see, for example, Kane, Curr.Opin. Biotechnol. 6:494, 1995, Makrides, Microbiol. Rev. 60:512, 1996,and Brown (Ed.), Molecular Biology LabFax (BIOS Scientific Publishers,Ltd. 1991), which provides a codon usage table on pages 245-253).

The present invention contemplates the use of “anionic spacer peptide”as that term is defined above. As described below, an illustrativeanionic spacer peptide has the amino acid sequence of HEAEPEAEPIM (SEQID NO: 27) where the methionine residue can be used as a cleavage site.Similar naturally occurring examples of anionic spacer peptides includeEAEPEAEP (SEQ ID NO: 28), EAKPEAEP (SEQ ID NO: 29), EAEPKAEP (SEQ ID NO:30), EAESEAEP (SEQ ID NO: 31), EAELEAEP (SEQ ID NO: 32), EPEAEP (SEQ IDNO: 33) and EAEP (SEQ ID NO: 34) (Casteels-Josson, et al. EMBO J.,12:1569-1578, 1993). Additional anionic spacer peptides are suitable foruse in producing cationic peptides such as doubles or other combinationsof those illustrated above. When designing an anionic spacer peptide forexpression of a particular cationic peptide in the multi-domain proteinconcept, the following criteria should be borne in mind: the negativecharge of the anionic spacer peptide should substantially reduce thepositive charge of the cationic peptide in the multi-domain fusionproteins, a cleavage point must be present at which the multi-domainprotein will be cleaved to give monomers of the desired peptide, and theanionic spacer peptide is preferably smaller than the cationic peptide.Such fusion proteins can be designed with alternating units of cationicpeptide and anionic spacer peptide. Such a configuration, however, isnot required. Any sequence of cationic peptide and anionic spacerpeptide is acceptable, as long as the cumulative charge of theconcatomer in the multidomain protein will not effect its expression inhost cells.

In the examples described herein, a cellulose binding domain (CBD)carrier protein was used to illustrate methods for producing cationicpeptides. Additional suitable examples of carrier proteins include, butare not limited to, glutathione-S-transferase (GST), Staphylococcusaureus protein A, two synthetic IgG-binding domains (ZZ) of protein A,outer membrane protein F, β-galactosidase (lacZ), and various productsof bacteriophage λ and bacteriophage T7. From the teachings providedherein, it is apparent that many other proteins may be used as carriers.As shown by the use of the CBD fragment, an entire carrier protein neednot be used, as long as it is highly expressed in the host cell. For thesake of simplicity and economics, suitable carrier proteins should be assmall as possible, around 100 amino acid residues or preferably less. Incertain cases, it is desirable to use a carrier protein that eitherlacks cysteine residues or that contains no more than one cysteineresidue. It is also desirable to avoid methionine residues except in thecleavage site if CNBr reagent is to be used to release the linkedpeptide.

To facilitate isolation of the cationic peptide sequence, amino acidssusceptible to cleavage can be used to bridge the carrier protein, acationic peptide moiety, and an anionic spacer peptide moiety in themulti-domain protein. The determination and design of the amino acidsequence of the cleavage site is highly dependent on the strategy ofcleavage and the amino acid sequence of the cationic peptide, anionicspacer peptide and carrier protein. The removal of the cationic peptidecan be accomplished through any known chemical or enzymatic cleavagesspecific for peptide bonds. Chemical cleavages include (R. A. Jue & R.F. Doolittle, Biochemistry, (1985) 24: 162-170; R. L. Lundblad, ChemicalReagents for Protein Modification (CRC Press, Boca Raton, Fla.; 1991),Chapter 5.), but are not limited to those treated by cyanogen bromidecleavages at methionine (Met↓), N-chlorosuccinimide or o-iodosobenzoicacid at tryptophan (Trp↓), hydroxylamine at asparaginyl-glycine bonds(Asn↓Gly), or low pH at aspartyl-proline bonds (Asp↓Pro). Alternatively,there are a vast number of proteases described in the literature but themajority have little specificity for a cleavage site. Enzymaticcleavages which can be performed include, but are not limited to thosecatalyzed by Factor Xa, Factor XIIa, thrombin, enterokinase,collagenase, Staphylococcus aureus V8 protease (endoproteinase Glu-C),endoproteinase Arg-C, endoproteinase Lys-C, chymotrypsin or trypsin.

To express a cationic peptide gene, a nucleic acid molecule encoding thepeptide must be operably linked to regulatory sequences that controltranscription and translation (expression) in an expression vector andthen introduced into a host cell. In addition, expression vectors caninclude a marker gene which is suitable for selection of cells thatcarry the expression vector.

The expression vectors of the present invention comprise nucleic acidmolecules encoding multi-domain fusion proteins with more than one copyof a cationic peptide gene. As can be shown, multi-domain fusionproteins having a carrier protein domain, an anionic spacer peptidecomponent, and a cationic peptide component may include from two to morethan 30 copies, of a cationic peptide gene. Multi-domain fusion proteinsthat lack an anionic spacer peptide component, but contain a carrierprotein domain and a cationic peptide component include two to fourcopies of a cationic peptide gene. Moreover, multi-domain fusionproteins that lack a carrier protein domain, but include both anionicspacer peptide and cationic peptide components may include from five tomore than 20 copies of a cationic peptide gene.

Preferably, cationic peptides are produced in prokaryotic host cells.Suitable promoters that can be used to express polypeptides in aprokaryotic host are well-known to those of skill in the art and forexample include T4, T3, SP6 and T7 promoters recognized by specificphage RNA polymerases, the int, P_(R) and P_(L) promoters ofbacteriophage lambda, the trp, recA, heat shock, lacUV5, tac,Ipp-lacSpr, phoA, lacP, tacP, trcp, srpP, araP, and lacZ promoters of E.coli, promoters of B. subtilis, the promoters of the bacterio-phages ofBacillus, Streptomyces promoters, the bla promoter of the cat promoter.Prokaryotic promoters have been reviewed by Glick, J. Ind. Microbiol.1:277, 1987, Watson et al., Molecular Biology of the Gene, 4th Ed.(Benjamin Cummins 1987), and by Ausubel et al. (1995).

Preferred prokaryotic hosts include E. coli, Bacillus subtilus, andStaphylococcus aureus. Suitable strains of E. coli include BL21(DE3),BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF′, DH5IMCR,DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38,RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for example, Brown(Ed.), Molecular Biology Labfax (Academic Press 1991)). Suitable strainsof Bacillus subtilus include BR151, YB886, MI119, MI120, and B170 (see,for example, Hardy, “Bacillus Cloning Methods,” in DNA Cloning: APractical Approach, Glover (Ed.) (IRL Press 1985)). An illustrativestrain of Staphylococcus aureus is RN4220 (Kreiswirth et al., Nature305:709, 1983). The present invention does not require the use ofbacterial strains that are protease deficient.

An expression vector can be introduced into host cells using a varietyof standard techniques including calcium phosphate transfection,microprojectile-mediated delivery, electroporation, and the like.Methods for introducing expression vectors into bacterial cells areprovided by Ausubel (1995). Methods for expressing proteins inprokaryotic hosts are well-known to those of skill in the art (see, forexample, Williams et al., “Expression of foreign proteins in E. coliusing plasmid vectors and purification of specific polyclonalantibodies,” in DNA Cloning 2: Expression Systems, 2nd Edition, Gloveret al. (eds.), page 15 (Oxford University Press 1995); and Georgiou,“Expression of Proteins in Bacteria,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc.1996)).

Cationic peptides can also be expressed in recombinant yeast cells.Promoters for expression in yeast include promoters from GAL1(galactose), PGK (phosphoglycerate kinase), ADH (alcohol dehydrogenase),AOX1 (alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like.Many yeast cloning vectors have been designed and are readily available.These vectors include YIp-based vectors, such as YIp5, YRp vectors, suchas YRp17, YEp vectors such as YEp13 and YCp vectors, such as YCp19. Oneskilled in the art will appreciate that there are a wide variety ofsuitable vectors for expression in yeast cells.

The baculovirus system provides an efficient means to express cationicpeptide genes in insect cells. Suitable expression vectors are basedupon the Autographa californica multiple nuclear polyhedrosis virus(AcMNPV), and contain well-known promoters such as Drosophila heat shockprotein (hsp) 70 promoter, Autographa californica nuclear polyhedrosisvirus immediate-early gene promoter (ie-1) and the delayed early 39Kpromoter, baculovirus p10 promoter, and the Drosophila metallothioneinpromoter. Suitable insect host cells include cell lines derived fromIPLB-Sf-21, a Spodoptera frugiperda pupal ovarian cell line, such as Sf9(ATCC CRL 1711), Sf21AE, and Sf21 (Invitrogen Corporation; San Diego,Calif.), as well as Drosophila Schneider-2 cells. Established techniquesfor producing recombinant proteins in baculovirus systems are providedby Bailey et al., “Manipulation of Baculovirus Vectors,” in Methods inMolecular Biology, Volume 7: Gene Transfer and Expression Protocols,Murray (ed.), pages 147-168 (The Humana Press, Inc. 1991), by Patel etal., “The baculovirus expression system,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al. (eds.), pages 205-244 (OxfordUniversity Press 1995), by Ausubel (1995) at pages 16-37 to 16-57, byRichardson (ed.), Baculovirus Expression Protocols (The Humana Press,Inc. 1995), and by Lucknow, “Insect Cell Expression Technology,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 183-218 (John Wiley & Sons, Inc. 1996). Established methods forisolating recombinant proteins from a baculovirus system are describedby Richardson (ed.), Baculovirus Expression Protocols (The Humana Press,Inc. 1995).

The recombinant host cells are cultured according to means known in theart to achieve optimal cell growth. In the case of recombinant bacterialhosts, preferably E. coli, the bacteria are introduced into a suitableculture medium containing nutrient materials that meet growthrequirements. After inoculation, the bacteria continue to divide andgrow until reaching a concentration, saturation density. For example,shake flask fermentation may require 15-17 hours at 30° C. to reach thispoint. Then the bacteria are diluted 1:3 in fresh medium and allowed togrow to mid- or late-exponential phase, at which time synthesis of thecationic peptide is induced. There are several methods of inducing thebacteria to synthesize the relevant recombinant proteins. Suitableinduction conditions will vary with the strain of E. coli and theplasmid it contains. For example, in the case of temperature-dependentinduction, the induction is obtained by raising the temperature to 42°C. and maintaining it from about 1 to about 5 hours at a preferred pHrange of 6.5-7.2. When the expression of the desired gene reachesoptimum levels the bacteria are harvested and the cells are eitherfrozen or continue through the recovery process.

b. Illustrative Vectors Having a Nucleotide Sequence Encoding a CarrierProtein-Cationic Peptide

As described in detail in the examples, plasmids were constructed thatcontained illustrative carrier protein genes. Briefly, plasmid vectorpET21a(+), a T7 expression plasmid (Novagen Corporation, USA), was usedas the core plasmid for initial studies (FIG. 1A). Plasmid pET-CBD180(see Shpigel et al., Biotech. Bioeng. 65:17-23, 1999) was used as thesource for the gene encoding the cellulose binding domain (CBD) carrierprotein (FIG. 1B). A PCR reaction was designed to amplify a fragmentcontaining the CBD180 gene from pET-CBD180 as a 646 bp fragment (FIG.1C). A BamHI restriction site (GGATCC) was incorporated at the 3′-end ofthe CBD180 PCR fragment. The BglII or XbaI sites of pET-CBD180 and BamHIwere used to cleave the PCR fragment and two fragments were separatelyligated into pET21a(+) resulting in two plasmids pET21CBD-B andpET21CBD-X, respectively (FIG. 1D). Plasmid pET21CBD-X contains lacOfrom pET21a(+), which improves the regulation of the T7 expressionsystem. Both plasmids contain a stop codon downstream of BamHI to allowexpression of CBD180 protein. A T7 expression system was prepared in E.coli MC4100 based on pGP1-2, which carries the T7 RNA polymerase geneunder a λ_(R) promoter controlled by cI857 thermo-sensitive repressor.CBD180 protein was expressed at high levels in both systems. PlasmidpET21CBD-X was used for subsequent studies.

Indolicidin is a natural 13-amino acid antimicrobial cationic peptidepresent in the cytoplasmic granules of bovine neutrophils and has aunique composition consisting of 39% tryptophan and 23% proline. Initialstudies used two cationic peptides derived from modifications ofindolicidin, MBI-11 peptide (I L K K W P W W P W R R K) (SEQ ID NO: 35)and MBI-11B7 peptide (I L R W P W W P W R R K) (SEQ ID NO: 36), asdescribed by Falla et al., WO 97/08199, and by Fraser et al., WO97/07745. A gene encoding the indolicidin-type cationic peptide MBI-11was synthesized with BamHI and HindIII cloning sites, fused to CBD180carrier protein and expressed. The level of expression was high andequal to that of CBD180 alone. Next, a tandem of two MBI-11 genes (2x11)was fused to CBD180, and again high expression was achieved. In order toincrease the ratio of cationic peptide to carrier protein, the 177 aminoacids of CBD180 were truncated to 96 amino acids, and this version ofthe carrier protein, designated CBD96, was used as a new carrierprotein. The DNA fragment carrying CBD96 was prepared by PCR, usingpET-CBD180 as a template, and cloned into pET21a(+) resulting in plasmidpET21CBD96. Both single and double copies of the MBI-11 gene were fusedto CBD96 and expressed at high levels. Then poly genes containing up toten MBI-11 units were prepared. However, expression was only achievedwith a fusion protein containing four MBI-11 genes in tandem. A dramaticdecrease in expression was encountered when the number of genes exceededthree (FIGS. 2 and 3).

c. Illustrative Vectors Having a Nucleotide Sequence Encoding a CarrierProtein-Cationic Peptide with Anionic Spacers

In another approach, vectors were constructed comprising multi-domainfusion proteins with small anionic peptide spacers between the cationicpeptide domains. This method of construction of multi-domain genesallows the polymerization of any cationic peptide gene without changingits amino acid sequence. In initial studies, the MBI-11B7 cationicpeptide was used.

Three distinct DNA cassettes specifying MBI-11B7 cationic peptide genesand a negatively charged peptide spacer were synthesized: 11B7-poly,anionic spacer, and 2x11B7-last (FIG. 4), and cloned into appropriateplasmid vectors. Cassettes of 11B7-poly and spacer were linked togetherresulting in the 11B7poly-spacer cassette. The anionic spacer peptideand cationic peptide genes were separated by codons for Met to createsites for cleavage by cyanogen bromide (CNBr). Two codons, specifyingAla and a stop codon, were linked to the last 2x11B7 gene. The2x11B7-last cassette was then cloned downstream of the gene encodingCBD96 in pET21CBD96 (FIG. 5) resulting in plasmid pET21CBD96-2x11B7.This plasmid was later used in the construction of several fusedmulti-domain genes. The 11B7poly-spacer cassette was used in a serialcloning procedure which allowed polymerization of 11B7 genes intomulti-domain fusion CBD96-spacer-poly11B7 proteins in the pET21CBD96expression system (FIG. 6). All multi-domain constructs containing ncopies (where n=3 to 30) of MBI-11B7 genes and (n-2) spacers wereexpressed at high levels. Examples of expression are shown in FIG. 7. Inorder to accelerate the serial cloning procedure a polymerizationcassette containing five 11B7 domains and five anionic spacer domainswas prepared and used for construction of multidomain genes containingmore than fifteen 11B7 domains (i.e., 20 copies, 25, 30, etc.). Thiscassette has an anionic spacer domain at the end followed by a stopcodon. Use of this cassette allowed construction of CBD96-basedmulti-domain systems containing equal numbers of 11B7 and spacerdomains.

d. Illustrative Vectors Having a Nucleotide Sequence Encoding a CationicPeptide with Anionic Spacers, but Lacking a Carrier Protein

One series of the multi-domain proteins comprises n times MBI-11B7peptides and n-2 anionic spacer peptides. When n=5, the molecular weightof the multi-domain protein equals 13.46 kDa, which should besufficiently large for expression in E. coli. DNA fragments containingmulti-domain genes of approximately this size were excised from relevantplasmids using restriction endonucleases NdeI and HindIII and fused intoplasmid containing specifically designed leader 11B7 domain. In E. coli,the first methionine in all proteins is translated as formyl-methioninewhich cannot be cleaved by CNBr. Accordingly, the carrier-freemulti-domain proteins were modified in such a way that the first domainbegins with M-T-M amino acids, allowing CNBr to cleave the first peptideat the second methionine and release authentic peptide. The relevantportions of plasmids pET21-3S-5x11B7 and pET21-5S-7x11B7 are shown inFIG. 8. All of the carrier-free multi-domain constructs containing from5 to 14 copies of MBI-11B7 genes were expressed at high levels as shownin FIG. 7. In the same way, constructs were prepared containing an equalnumber of 11B7 and anionic spacer domains with a spacer sequence at theend. They were also expressed at high levels. The theoretical yield ofthe MBI-11B7 peptide, within experimentally obtained multi-domainproteins, can be seen in Table 2.

The invention also provides an additional example of anotherantimicrobial cationic peptide (MBI-26), twice the size of the peptidedescribed above (MBI-11B7), consisting of 26 amino acids, where seven ofthem are basic amino acids. This peptide was artificially designed by afusion between selected sequences of the natural antimicrobial cationicpeptides cecropin and melittin. In the present invention, the last aminoacid serine at the carboxy end was replaced with a methionine residue,which was used for release of the peptide from the multi-domain protein.The production of this peptide was obtained by recombinant synthesis inhost cells, using the multi-domain protein method, as described abovefor MBI-11B7 peptide. Details are provided in Example 8. TABLE 2 SUMMARYOF SUCCESSFULLY EXPRESSED CONSTRUCTS* AND THEIR THEORETICALMBI-11B7CATIONIC PEPTIDE RATIO IN THE MULTI-DOMAIN PROTEINS, WITH ANDWITHOUT CARRIER PROTEIN Multi-domain % Cationic Peptide per Protein MassMulti-domain Protein Construct (Da) (Da/Da) With Carrier Protein — —pET21CBD-11B7 21,249 8.9 pET21CBD-2x11B7 23,142 16.5 pET21CBD96-11B712,697 15.0 pET21CBD96-2x11B7 14,590 26.1 pET21CBD96-1S-3x11B7 17,71832.3 pET21CBD96-2S-4x11B7 20,845 36.6 pET21CBD96-3S-5x11B7 23,973 39.8pET21CBD96-4S-6x11B7 27,101 42.2 pET21CBD96-5S-7x11B7 30,228 44.2pET21CBD96-6S-8x11B7 33,356 45.8 pET21CBD96-7S-9x11B7 36,484 47.1pET21CBD96-8S-10x11B7 39,612 48.2 pET21CBD96-9S-11x11B7 42,739 49.1pET21CBD96-10S-12x11B7 45,867 49.9 pET21CBD96-11S-13x11B7 48,995 50.6pET21CBD96-12S-14x11B7 52,122 51.3 pET21CBD96-13S-15x11B7 55,250 51.8pET21CBD96-188-20x11B7 70,888 53.9 pET21CBD96-23S-25x11B7 86,527 55.1pET21CBD96-28S-30x11B7 102,162 56.1 With equal spacers number — —pET21CBD96-5S-5x11B7 26,282 36.3 pET21CBD96-10S-10x11B7 41,921 45.5pET21CBD96-15S-15x11B7 57,559 48.9 Without Carrier Protein — —pET21-3s-5x11B7-F 13,692 69.7 pET21-4s-6x11B7-F 16,820 68.1pET21-5s-7x11B7-F 19,947 67.0 pET21-6s-8x11B7-F 23,075 66.2pET21-7s-9x11B7-F 26,203 65.6 pET21-8s-10x11B7-F 29,330 65.1pET21-9s-11x11B7-F 32,458 64.7 pET21-10s-12x11B7-F 35,586 64.4pET21-11s-13x11B7-F 38,713 64.1 pET21-12s-14x11B7-F 41,841 63.9pET21-19s-21x11B7-F 63,735 61.9 With equal spacers number — —pET21-6s-6x11B7-F 19,129 59.9 pET21-11s-11x11B7-F 34,767 60.4pET21-16s-16x11B7-F 50,405 60.6*Examples of the expression can be seen in FIG. 7.

4. Purification and Assay of Cationic Peptides Produced by RecombinantHost Cells

General techniques for recovering protein produced by a recombinant hostcell are provided, for example, by Grisshammer et al., “Purification ofover-produced proteins from E. coli cells,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al. (eds.), pages 59-92 (OxfordUniversity Press 1995), Georgiou, “Expression of Proteins in Bacteria,”in Protein Engineering: Principles and Practice, Cleland et al. (eds.),page 101 (John Wiley & Sons, Inc. 1996), Richardson (ed.), BaculovirusExpression Protocols (The Humana Press, Inc. 1995), and by Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163 (Wiley-Liss, Inc. 1996). Variations in cationic peptideisolation and purification can be devised by those of skill in the art,including, for example, affinity chromatography, size exclusionchromatography, ion exchange chromatography, HPLC and the like (see, forexample, Selsted, “HPLC Methods for Purification of AntimicrobialPeptides,” in Antibacterial Peptide Protocols, Shafer (ed.) (HumanaPress, Inc. 1997)). Particular purification methods are described below.

The present invention provides a novel, scaleable, cost-effectivepurification process for recombinant production of cationic peptides inhost cells. The multi-domain fused polypeptide forms an insolublecomplex in E. coli called the inclusion body. After the bacteria aremechanically disrupted, these inclusion bodies can be separated from thesoluble components of the cell, according to means known in the art suchas filtration or precipitation. Host impurities can be removed usingsolvents such as detergent (Triton X-100), enzyme (lysozyme, DNAse) andsalt.

Cationic peptides can be released from anionic spacer peptides andcarrier protein (such as truncated CBD) using standard techniques. Ifmethionine residues have been included at desired cleavage points, forexample, chemical cleavage with cyanogen bromide (CNBr) reagent in anacidic environment can be used. The reaction can be performed in 70%formic acid or 70% formic acid and 0.1 N HCl or 70% TFA (trifluoroaceticacid). At the end of the reaction, which can last 4-15 hours, thereaction mixture is diluted in water, preferably 15 times the volume ofthe reaction mixture, and then dried. At this stage, the carboxylterminus of the cationic peptide is present as homoserine lactone.

The isoelectric point of polycationic peptides is very high (10.5-12.5)which enables the development of a very unique purification processusing an anion exchange chromatography column under unusual conditions.Almost any anion exchange resin coupled to weak or strong cation ligand,particularly those used for industrial purpose to purify proteins,peptides, carbohydrates and nucleic acids, can be used in the followingpurification process for cationic peptides. This procedure requires theuse of only one chromatography step to obtain 95% purification. Theadvantages of this chromatography are that it is short, fast, does notrequire high pressure equipment and can be performed without organicsolvents. The preferred procedure relies on dissolving the driedcleavage materials in 7-8 M urea (alternatively in 50% ethanol or water)and loading them onto an anion exchange column. At this stage, the pH ofthe loading sample is acidic (pH 2-3). The column is previously washedwith two column volumes of 0.5-1 M NaOH and one short wash in water to aconductivity of less than 10 mS, preferably less than 1 mS, detected atthe exit of the column. If the dried cleavage materials have beendissolved in 8 M urea, one column wash with 8 M urea before loading ispreferred. The cationic peptide, in contrast to the impurities, passesthrough the column whereas the impurities are bound to the resin andthus separated (FIG. 9). At this stage, the carboxy terminus of thecationic peptide has been converted and appears as homoserine. Inaddition, the pH of the cationic peptide sample has changed from acidicto basic (above pH 11).

If the dried cleavage materials loaded on the anion exchange column arein the presence of 7-8 M urea, the flow through purified peptide will bein urea solution, which can be separated and further purified byhigh-throughput reverse phase chromatography using the perfusivesupports Poros 20 or 50 R-2 resin (PerSeptive Biosystems Inc.). For massproduction, Poros 50 is preferred due to better flow and the fact thatthe use of high pressure equipment is avoided.

Another more common, but more expensive procedure can be performedaccording to means known in the art, such as reverse phasechromatography where the dried cleavage materials may be dissolved inwater or 0.1% TFA and loaded onto a C8 or C18 column using the RP-HPLCtechnique. However, this method requires high pressure equipment andorganic solvents and results in a cationic peptide with a C-terminalhomoserine lactone.

In the studies described above, the recombinant cationic peptideMBI-11B7 was obtained from a multi-domain construct. As a result, CNBrcleavage causes the formation of a homoserine lactone residue at thecarboxy end which may be easily converted to homoserine by raising thepH. This carboxy terminus is different from the bactericidal amidatedchemical synthetic MBI-11B7CN. Hence, the antimicrobial activity wascompared between chemically and recombinantly synthesized cationicpeptide.

There are various in vitro methods for determining the activity of acationic peptide, including an agarose dilution MIC assay, a brothdilution, time-kill assay, or equivalent methods (see, for example,Shafer (ed.), Antibacterial Peptide Protocols (Humana Press, Inc.1997)). Antibiotic activity is typically measured as inhibition ofgrowth or killing of a microorganism or a microorganism-infected cell.

For example, a cationic peptide is first dissolved in Mueller Hintonbroth supplemented with calcium and magnesium, and then this solution ismixed with molten agarose. Other broth and agars may be used as long asthe peptide can freely diffuse through the medium. The agarose is pouredinto petri dishes or wells and allowed to solidify, and a test strain isapplied to the agarose plate. The test strain is chosen, in part, basedon the intended application of the peptide. Plates are incubatedovernight and inspected visually for bacterial growth. A minimuminhibitory concentration (MIC) of a cationic peptide is the lowestconcentration of peptide that completely inhibits growth of theorganism. Peptides that exhibit acceptable activity against the teststrain, or group of strains, typically having an MIC of less than orequal to 16 μg/ml, can be subjected to further testing.

Alternatively, time kill curves can be used to determine the differencesin colony counts over a set time period, typically 24 hours. Briefly, asuspension of organisms of known concentration is prepared and acationic peptide is added. Aliquots of the suspension are removed at settimes, diluted, plated on medium, incubated, and counted. MIC ismeasured as the lowest concentration of peptide that completely inhibitsgrowth of the organism.

Cationic peptides may also be tested for their toxicity to normalmammalian cells. An exemplary assay is a red blood cell (RBC)(erythrocyte) hemolysis assay. Briefly, in this assay, red blood cellsare isolated from whole blood, typically by centrifugation, and washedfree of plasma components. A 5% (v/v) suspension of erythrocytes inisotonic saline is incubated with different concentrations of cationicpeptide. After incubation for approximately one hour at 37° C., thecells are centrifuged, and the absorbance of the supernatant at 540 nmis determined. A relative measure of lysis is determined by comparisonto absorbance after complete lysis of erythrocytes using NH₄Cl orequivalent (establishing a 100% value). A peptide with less than 10%lysis at 100 g/ml is suitable. Preferably, the cationic peptide inducesless than 5% lysis at 100 g/ml. Cationic peptides that are not lytic, orare only moderately lytic, are desirable and suitable for furtherscreening. In vitro toxicity may also be assessed by measurement oftoxicity towards cultured mammalian cells.

Additional in vitro assays may be carried out to assess the therapeuticpotential of a cationic peptide. Such assays include peptide solubilityin formulations, pharmacology in blood or plasma, serum protein binding,analysis of secondary structure, for example by circular dichroism,liposome permeabilization, and bacterial inner membranepermeabilization.

In the present case, the antimicrobial activities of MBI-11B7CN,MBI-11B7HSL (homoserine lactone form) and MBI-11B7HS (homoserine form)were tested against various gram-negative and gram-positive strains,including antibiotic resistant strains. The assay was performed asdescribed in “Methods for Dilution Antimicrobial Susceptibility Testsfor Bacteria That Grow Aerobically-Fourth Edition; Approved Standard”NCCLS document M7-A4 (ISBN 1-56238-309-4) Vol. 17, No. 2 (1977).Determination of the minimum inhibitory concentration (MIC) of thepeptides, demonstrated that MBI-11B7HSL and MBI-11B7HS peptides maintainsimilar bactericidal activity to the amidated MBI-11B7CN peptide. SeeTable 3 in Example 13.

Cationic peptides can also be tested in vivo for efficacy, toxicity andthe like. The antibiotic activity of selected peptides may be assessedin vivo for their ability to ameliorate microbial infections using avariety of animal models. A cationic peptide is considered to betherapeutically useful if inhibition of microorganism growth, comparedto inhibition with vehicle alone, is statistically significant. Thismeasurement can be made directly from cultures isolated from body fluidsor sites, or indirectly, by assessing survival rates of infectedanimals. For assessment of antibacterial activity, several animal modelsare available, such as acute infection models including those in which(a) normal mice receive a lethal dose of microorganisms, (b) neutropenicmice receive a lethal dose of microorganisms, or (c) rabbits receive aninoculum in the heart, and chronic infection models. The model selectedwill depend in part on the intended clinical indication of the cationicpeptide.

As an illustration, in a normal mouse model, mice are inoculated ip oriv with a lethal dose of bacteria. Typically, the dose is such that90-100% of animals die within two days. The choice of a microorganismstrain for this assay depends, in part, upon the intended application ofthe cationic peptide. Briefly, shortly before or after inoculation(generally within 60 minutes), cationic peptide is injected in asuitable formulation buffer. Multiple injections of cationic peptide maybe administered. Animals are observed for up to eight dayspost-infection and the survival of animals is recorded. Successfultreatment either rescues animals from death or delays death to astatistically significant level, as compared with non-treatment controlanimals.

In vivo toxicity of a peptide can be measured by administration of arange of doses to animals, typically mice, by a route defined in part bythe intended clinical use. The survival of the animals is recorded andLD₅₀, LD₉₀₋₁₀₀, and maximum tolerated dose (MTD) can be calculated toenable comparison of cationic peptides.

Low immunogenicity of the cationic peptide is also a preferredcharacteristic for in vivo use. To measure immunogenicity, peptides areinjected into normal animals, generally rabbits. At various times aftera single or multiple injections, serum is obtained and tested forantibody reactivity to the peptide analogue. Antibodies to peptides maybe identified by ELISA, immunoprecipitation assays, Western blots, andother methods (see, generally, Harlow and Lane, Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory Press, 1988)).

Expression vectors comprising the multi-domain fusion proteins describedherein can be used to produce multi-domain fusion protein representingmore than 25% of the total protein of a recombinant host cell. Since themulti-domain fusion proteins comprise multiple copies of a cationicpeptide gene, the cationic peptide component of a fusion protein can bepractically attained as more than 50% of the fusion protein.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

EXAMPLE 1 Construction of Plasmids PET21CBD-X(B) AND PET21CBD96

Plasmid vector pET21a(+) (Novagen Corporation, USA), a T7 expressionplasmid, was used as the core plasmid for all expression systems (FIGS.1A and 10). The cellulose binding domain (CBD) from Clostridiumcellulovorans was selected as a carrier protein for expression ofantibacterial cationic peptides. Plasmid pET-CBD180 (Shpigel et al.,supra) was used as the starting material (FIGS. 1B and 10). Restrictionenzymes except VspI and NsiI (Promega Corporation, USA), T4 DNA ligaseand Taq polymerase were purchased from Pharmacia Biotech. The relevantpart of CBD, including the T7 promoter of pET-CBD180, was amplified byPCR using 25 pmol each of each of the primers GCGT CCGG CGTA GAGG ATCG(SEQ ID NO:1) and CCGG GATC CAAT GTTG CAGA AGT AG (SEQ ID NO:2), 2 U ofTaq DNA polymerase, corresponding reaction buffer (10× PCR reactionbuffer: 500 mM KCl, 15 mM MgCl₂, 100 mM Tris-HCl, pH 9), 0.2 mM dNTPs(dATP, dGTP, dTTP and dCTP, Pharmacia Biotech) and 20 ng ofheat-denatured pET-CBD180. PCR was performed in MJ-Research PTC-100Thermo-cycler in 50 μl reaction volume and 30 cycles of 94° C., 30 sec.;55° C., 30 sec. and 72° C., 30 sec. A BamHI restriction site (GGATCC)was incorporated at the 3′-end of the cbd gene to allow it to be clonedinto pET21a(+). The BglII (AGATCT) or XbaI (TCTAGA) sites alreadypresent on pET-CBD180 were used to cut the 5′-end of the PCR fragment.One μg of PCR product was digested in a 100 μl reaction containing 1.5×OPA (Pharmacia Biotech assay buffer One-Phor-AII is supplied at 10×concentration: 100 mM Tris-acetate, pH 7.5; 100 mM magnesium acetate and500 mM potassium acetate) and 10 U of BamHI and 10 U of HindIII. PlasmidpET21a(+) was digested in the same way, in 2×50 μl reactions eachcontaining 0.25 μg of plasmid DNA, 1.5× OPA and 2 U of BamHI and HindIIIeach. Reactions were stopped by phenol/CHCl₃ extraction and ethanolprecipitation. The resultant DNA pellets of digested pET21a(+) andrelevant cbd and cbd96 inserts were dissolved in 8 μl of water andmixed, then 2 μl of 10 mM ATP, 2 μl of 10× OPA and 2 U of T4 DNA ligasewere added and reactions were incubated at 10° C. for 1 hour. Then 2 μlof each ligation mixture were used to electroporate 40 μl of E. coli XL1Blue (Promega Corporation) using a sterile Gene Pulser cuvette (0.2 cmelectrode gap) and Gene Pulser electroporator apparatus (Bio-RadLaboratories) set to 2.5 kV, 200 ohms and 250 μF. After anelectroporation pulse, 1 ml of TB media (Maniatis, et al., MolecularCloning: A Laboratory Manual, 2^(nd Ed)., (Cold Spring Harbor LaboratoryPress 1989) was added to the cell suspension and bacteria were incubatedfor 1 hour at 37° C. with rigorous shaking. Then 10, 50 and 100 μl ofcell suspension were plated on MacKonkey agar (BBL, Becton Dickinson andCompany, USA) plates with 100 μg/ml of Ampicillin and incubatedovernight at 37° C. The next day, several colonies were transferred to 2ml of TB and cultivated at 37° C. with vigorous shaking overnight. Thenplasmid DNA was isolated and analyzed, including DNA sequencing bymethods known to those skilled in the art. Positive clones containedplasmids pET21CBD-B or pET21CBD-X, respectively (FIG. 10). PlasmidpET21CBD-X contains lacO, which improves the regulation of the T7expression system. Both plasmids contain a stop codon downstream ofBamHI to allow expression of CBD180 protein. A T7 expression system wasprepared in E. coli MC4100F (Strain MC4100F was prepared by mating E.coli XL1Blue and E. coli MC4100; ATCC Number 35695) based on pETvariants and pGP1-2, which carries the T7 RNA polymerase gene under a λRpromoter controlled by cI857 thermo-sensitive repressor (Tabor andRichardson, Biochemistry 82:1074, 1985). CBD180 protein was expressed athigh levels in both systems. Plasmid pET21CBD-X was used for subsequentwork.

Plasmid pET21CBD96 (FIG. 5) was prepared using the same PCR conditionsand cloning procedures. In this experiment the carrier protein CBD180was truncated to about 96 amino acids. Therefore a pair of PCR primersGCGT CCGG CGTA GAGG ATCG (SEQ ID NO:3) and ATAT GGAT CCAG ATAT GTAT CATAGGTT GATG TTGG GC (SEQ ID NO:4) was used to prepare the relevant DNAfragment encoding cbd96 (FIG. 5), which was then cloned into pET21a(+).Then again a T7 expression system was prepared in E. coli MC4100F basedon plasmids pET21CBD96 and pGP1-2 and protein CBD96 was expressed athigh levels. pET21CBD96 was used for most of the subsequent work.

EXAMPLE 2 Construction and Expression of CBD—MBI-11 Fusions

Sequences encoding all cationic peptides were designed from modifiedindolicidin, a natural anti-microbial peptide. Plasmids pET21CBD-X andpET21CBD96 (0.25 μg each) were digested with 2 U of BamHI and 2 U ofHindIII in 1.5× OPA in 50 μl reactions at 37° C. for 1 hour. In the sameway, a fragment encoding MBI-11 was digested (Example 4) using about 1μg of DNA and 25U of BamHI and HindIII each in a 100 μL reaction. Bothreactions were stopped by phenol/CHCl₃ (Sigma-Aldrich Canada Ltd.)extraction and ethanol precipitation. The resultant DNAs of each vectorand MBI-11 insert were dissolved in 8 μl of water and mixed, then 2 μlof 10 mM ATP, 2 μl of 10× OPA and 2 U of T4 DNA ligase were added andligation reactions were incubated at 10° C. for 1 hour. Then 2 μl ofeach ligation mixture was used to electroporate 40 μl of E. coli XL1Blue using sterile Gene Pulser cuvettes (0.2 cm electrode gap) and GenePulser electroporator apparatus set to 2.5 kV, 200 ohms and 250 μF.After an electroporation pulse, 1 ml of TB media was added to the cellsuspension and bacteria were incubated for 1 hour at 37° C. withrigorous shaking. Then 10, 50 and 100 μl of cell suspension were platedon MacKonkey agar plates with 100 μg/ml of Ampicillin (Sigma-AldrichCanada Ltd.) and incubated overnight at 37° C. The next day, severalcolonies were transferred to 2 ml of TB and cultivated at 37° C. withvigorous shaking overnight. Then plasmid DNA was isolated and analyzed,including DNA sequencing by methods known to those skilled in the art.Positive clones contained MBI-11 fused to CBD180 or CBD96. Expressionstrains of E. coli MC4100F harboring plasmids pGP1-2 and pET21CBD-11 orpET21CBD96-11 respectively were prepared by electroporation. Finalstrains were incubated overnight in 2 ml TB at 30° C. with rigorousshaking and the next day 1 ml of cell suspension was diluted with theequal volume of fresh TB and cultivation temperature was increased to42° C. for a minimum of 2 hours. Samples of preinduced and induced cellswere analyzed by SDS-PAGE. The level of expression of the fusion proteincaring MBI-1 or 2× MBI-11 gene was high and equal to expression ofCBD180 or CBD96 alone.

EXAMPLE 3 Expression of CBD Fused Polycationic Peptide Tandem Domains

This experiment was designed to test how many peptide genes in tandemcan be fused to a carrier protein and expressed. It was necessary tocreate two DNA fragments encoding MBI-11, one for polymerization by DNAcloning and another one as the last gene in the tandem. Therefore, theoriginal DNA fragment encoding MBI-11 peptide with COOH end was modifiedin order to create the last gene in tandem (Example 4) and a new genewas designed for a specific cloning procedure, which allowedconstruction of multiple tandem peptide genes fused to CBD180 or CBD96carrier proteins genes (Example 4). The cloning procedure resulted inaddition of an extra isoleucine to the MBI-11 tandem sequences.Therefore in order to produce identical peptide molecules, an isoleucinecodon was also added to the last gene sequence. CNBr will be used tocleave the peptide from fusion proteins, which means that peptidemolecules would have a homoserine lactone on the end. Therefore the lastpeptide gene was also modified to have a methionine followed by twotyrosines at the end for CNBr cleavage in order to produce equivalentpeptide products.

CBD180 and CBD96 fused peptide polygenes of up to 10 units in tandemwere prepared. However good expression was only achieved with a fusioncontaining two and three MBI-11 domains and practically stopped when thenumber of peptide genes exceeded four. DNA synthesis and construction ofplasmids containing MBI-11 polymers is described in Example 4.

EXAMPLE 4 Synthesis and Modification of DNA Fragments Encoding CationicPeptides

The desired sequences were conventionally synthesized by thephosphoramidite method of oligonucleotide synthesis using the AppliedBiosystems Model 391 DNA Synthesizer with connected chemicals andprotocols. Desired oligonucleotides were used as templates in the PCRreaction to produce double stranded DNA suitable for DNA cloning.

A. Synthesis of the MBI-11 DNA Domain

An oligonucleotide TTTA ACGG GGAT CCGT CTCA TATG ATCC TGAA AAAA TGG (SEQID NO:5) CCGT GGTG GCCG TGGC GTCG TAAA TMG CTTG ATAT CTTG GTAC CTGC G(SEQ ID NO:6) was synthesized and used as a template for PCR usingprimers TTTA ACGG GGAT CCG TCTC ATAT G (SEQ ID NO:7) and TMG CTTG ATATCTTG GTAC CTGC G (SEQ ID NO:8). The PCR was performed in MJ-ResearchPTC-100 Thermo-cycler in a 50 μl reaction volume with 30 cycles of 94°C., 30 sec.; 50° C., 30 sec. and 72° C., 30 sec., 2 U of Taq DNApolymerase, corresponding reaction buffer (10× PCR reaction buffer: 500mM KCl, 15 mM MgCl₂, 100 mM Tris-HCl pH 9), 0.2 mM dNTPs (dATP, dGTP,dTTP and dCTP), 25 pmol of each primer and 50 pmol of templateoligonucleotide resulting in an 88 bp dsDNA MBI-11 fragment. DNA wasused for the cloning procedure described in Example 2.

B. Modification of MBI-11 Domain as the Last Domain in Tandem

PCR was used to modify the original DNA fragment encoding MBI-11 for useas the last gene in the tandem polypeptide gene. The originaloligonucleotide (A) was used as a template. The sense primer TTTA ACGGGGAT CCGT CTCA TATG (SEQ ID NO:9) was identical to that used in thesynthesis PCR reaction, but a new antisense primer CGCG MGC TTM TMT ACATMTT TTAC GACG CCAC GGCC ACCA CGGC (SEQ ID NO:10) was designed to modifythe end of the MBI-11 gene (for explanation, see Example 3). The PCR wasperformed in MJ-Research PTC-100 Thermo-cycler in a 50 μl reactionvolume with 30 cycles of 94° C. 30 sec., 51° C., 30 sec. and 72° C., 30sec., 2 U of Taq DNA polymerase, corresponding reaction buffer (10×PCRreaction buffer: 500 mM KCl, 15 mM MgCl₂, 100 mM Tris-HCl pH 9), 0.2 mMdNTPs (dATP, dGTP, dTTP and dCTP), 25 pmol of each primer and 50 pmol ofthe template oligonucleotide. The PCR product was then cloned as aBamHI-HindIII fragment into pBCKS(+) (Stratagene, USA) resulting inplasmid pBCKS-11. Modification was verified by DNA sequencing.

C. Synthesis of MBI-11 Fragment Designated for the PolymerizationCloning Procedure

An oligonucleotide CGCC AGGG TTTT CCCA GTCA CGAC GGAT CCGT CTCA TATGATCC TGM AAAA TGGC CGTG GTGG CCGT GGCG TCGT AAAA TTM TTGA ATTC GTCA TAGCTGTT TCCT GTGT GA (SEQ ID NO:11) was synthesized and used as a templatefor PCR using primers CGCC AGGG TTTT CCCA GTCA CGAC (SEQ ID NO:12) andTCAC ACAG GAAA CAGC TATG AC (SEQ ID NO:13). The PCR was performed inMJ-Research PTC-100 Thermo-cycler in a 50 μl reaction volume with 30cycles of 94° C., 30 sec., 51° C., 30 sec. and 72° C., 30 sec., 2U ofTaq DNA polymerase, corresponding reaction buffer (10× PCR reactionbuffer: 500 mM KCl, 15 mM MgCl₂, 100 mM Tris-HCl pH 9), 0.2 mM dNTPs(dATP, dGTP, dTTP and dCTP), 25 pmol of each primer and 50 pmol oftemplate oligonucleotide resulting in 114 bp dsDNA MBI-11-BE fragment.This fragment was cloned as a BamHI-EcoRI insert into vector pBCKS(+)resulting in pBCKS-11BE.

D. Polymerization Cloning Procedure

The copy of MBI-11 designed for the polymerization cloning procedure wascloned into pET21CBD96-11 resulting in pET21CBD96-2x11. pBCKS-11BE wasdigested with 2 U of BamHI and VspI in 2× OPA in 50 μl reactions at 37°C. for 1 hour and pET21CBD96-11 was digested with 2 U of BamHI and NdeIin 2× OPA in a 50 μl reaction at 37° C. for 1 hour. Reactions werestopped by phenol/CHCl₃ extraction and ethanol precipitation. Theresulting DNA pellets were dissolved in 8 μl of water each and mixed,then 2 μl of 10 mM ATP, 2 μl of 10× OPA and 2 U of T4 DNA ligase wereadded and reactions were incubated at 10° C. for 1 hour. Then 2 μl ofthe ligation mixture was used to electroporate 40 μl of E. coli XL1 Blueusing Gene Pulser cuvettes (0.2 cm electrode gap) and Gene Pulser(Bio-Rad Laboratories) set to 2.5 kV, 200 ohms and 250 μF. After anelectroporation pulse, 1 ml of TB media was added to the cell suspensionand bacteria were incubated 1 hour at 37° C. with rigorous shaking. Then10, 50 and 100 μl of cell suspension were plated on MacKonkey agarplates with 100 μg/ml of Ampicillin and incubated overnight at 37° C.The next day, several colonies were transferred to 2 ml of TB andcultivated at 37° C. with vigorous shaking overnight. Then plasmid DNAwas isolated and analyzed, including DNA sequencing by methods known tothose skilled in the art. Positive clones contained pET21CBD96-2x11. Theligation of compatible VspI and NdeI cohesive ends resulted toelimination of both restriction sites. At the same time, the insertionof the mbi-11be cassette introduced a new NdeI site, which allowedrepetition of the cloning procedure and insertion of another mbi-11be.This procedure could be repeated theoretically without limitation. Inthis particular case the serial cloning was repeated nine times andconstructs up to pET21CBD96-10x11 were prepared.

EXAMPLE 5 Synthesis of DNA Cassettes for Construction of Fused andUnfused Multi-Domain Expression Systems

A. Synthesis of MBI 2x11B7-Last Cassette

An oligonucleotide CGCC AGGG TTTT CCCA GTCA CGAC GGAT CCGT CTCA TATGATTC TGCG TTGG CCGT GGTG GCCG TGGC GTCG CAAA ATGA TTCT GCGT TGGC CGTGGTGG CCGT GGCG TCGC AAAA TGGC GGCC TAAG CTTC GATC CTCT ACGC CGGA CGC(SEQ ID NO:14) was synthesized and used as a template for PCR usingprimers CGCC AGGG TTTT CCCA GTCA CGAC (SEQ ID NO:15) and GCGT CCGG CGTAGAGG ATCG (SEQ ID NO:16). The PCR was performed in MJ-Research PTC-100Thermo-cycler in a 50 μl reaction volume with 30 cycles of 94° C., 30sec.; 55° C., 30 sec. and 72° C., 30 sec. 2 U of Taq DNA polymerase,corresponding reaction buffer (10×PCR reaction buffer: 500 mM KCl, 15 mMMgCl₂, 100 mM Tris-HCl pH 9), 0.2 mM dNTPs (dATP, dGTP, dTTP and dCTP),25 pmol of each primer and 50 pmol of template oligonucleotide resultingin 151 bp dsDNA MBI-11 fragment. The PCR product was purified byphenol/CHCl₃ extraction and ethanol precipitation. The resulting DNA wasdissolved in 100 μl 1× OPA, 20U of BamHI and 20U of HindIII and thereaction was incubated at 37° C. for 2 hours. The vector pBCKS(+) (0.25μg) was digested in the same way. Both reactions were stopped byphenol/CHCl₃ extraction and ethanol precipitation. The resultant DNAs ofeach vector and MBI-11 insert were dissolved in 8 μl of water and mixed,then 2 μl of 10 mM ATP, 2 μl of 10× OPA and 2 U of T4 DNA ligase wereadded and ligation reactions were incubated at 10° C. for 1 hour. Then 2μl of each ligation mixture was used to electroporate 40 μl of E. coliXL1 Blue using a sterile Gene Pulser cuvette (0.2 cm electrode gap) andGene Pulser electroporator apparatus set to 2.5 kV, 200 ohms and 250 μF.After an electroporation pulse, 1 ml of TB media was added to the cellsuspension and bacteria were incubated 1 hour at 37° C. with rigorousshaking. Then 10, 50 and 100 μl of cell suspension were plated onMacKonkey agar plates with 100 μg/ml of Ampicillin and incubatedovernight at 37° C. The next day, several colonies were transferred to 2ml of TB and cultivated at 37° C. with vigorous shaking overnight. Thenplasmid DNA was isolated and analyzed, including DNA sequencing bymethods known to those skilled in the art. The resulting plasmid waspBCKS-2x11B7. The insert was later recloned into pBCKS-V resulting inpBCKS-V-2x11B7.

B. Synthesis of MBI-11B7-Poly Cassette

An oligonucleotide CGCC AGGG TTTT CCCA GTCA CGAC GGAT CCGT CTCA TATGATTC TGCG TTGG CCGT GGTG GCCG TGGC GTCG CAAA ATGC ATM GCTT CGAT CCTCTACG CCGG ACGC (SEQ ID NO:17) was synthesized and used as a template forPCR using primers CGCC AGGG TTTT CCCA GTCA CGAC (SEQ ID NO:18) and GCGTCCGG CGTA GAGG ATCG (SEQ ID NO:19). The PCR was performed in MJ-ResearchPTC-100 Thermo-cycler in a 50 μl reaction volume with 30 cycles of 94°C., 30 sec.; 55° C., 30 sec. and 72° C., 30 sec., 2 U of Taq DNApolymerase, corresponding reaction buffer (10× PCR reaction buffer: 500mM KCl, 15 mM MgCl₂, 100 mM Tris-HCl pH 9), 0.2 mM dNTPs (dATP, dGTP,dTTP and dCTP), 25 pmol of each primer and 50 pmol of templateoligonucleotide resulting in a 112 bp dsDNA MBI-11 fragment. Theresulting DNA fragment was cloned into pTZ18R (Pharmacia Biotech) as aBamHI-HindIII fragment as described in paragraph (A) resulting inplasmid pTZ18R-11B7poly.

C. Synthesis of Anionic Spacer Cassette

An oligonucleotide CGCC AGGG TTTT CCCA GTCA CGAC GGAT CCGT CTAT GCAT GMGCGGA ACCG GAAG CGGA ACCG ATTA ATTA AGCT TCGA TCCT CTAC GCCG GACG C (SEQID NO:20) was synthesized and used as a template for PCR using primersCGCC AGGG TTTT CCCA GTCA CGAC (SEQ ID NO:21) and GCGT CCGG CGTA GAGGATCG (SEQ ID NO:22). The PCR was performed in MJ-Research PTC-100Thermo-cycler in a 50 μl reaction volume with 30 cycles of 94° C., 30sec.; 55° C., 30 sec. and 72° C., 30 sec., 2 U of Taq DNA polymerase,corresponding reaction buffer (10× PCR reaction buffer: 500 mM KCl, 15mM MgCl₂, 100 mM Tris-HCl pH 9), 0.2 mM dNTPs (dATP, dGTP, dTTP anddCTP), 25 pmol of each primer and 50 pmol of template oligonucleotideresulting in a 97 bp dsDNA MBI-11 fragment. The resulting DNA fragmentwas cloned into pBCKS-V as a BamHI-HindIII fragment as described inparagraph (A), resulting in plasmid pBCKS-V-S.

D. Synthesis of MBI-11B7-First Cassette

An oligonucleotide CGCC AGGG TTTT CCCA GTCA CGAC GGAT CCGT CTCA TATGACTA TGAT TCTG CGTT GGCC GTGG TGGC CGTG GCGT CGCA AAAT GCAT MGC TTCGATCC TCTA CGCC GGAC GC (SEQ ID NO:23) was synthesized and used as atemplate for PCR using primers CGCC AGGG TT TT CCCA GTCA CGAC (SEQ IDNO:24) and GCGT CCGG CGTA GAGG ATCG (SEQ ID NO:25). The PCR wasperformed in MJ-Research PTC-100 Thermo-cycler in a 50 μl reactionvolume with 30 cycles of 94° C., 30 sec.; 55° C., 30 sec. and 72° C., 30sec, 2 U of Taq DNA polymerase, corresponding reaction buffer (10× PCRreaction buffer: 500 mM KCl, 15 mM MgCl₂, 100 mM Tris-HCl, pH 9), 0.2 mMdNTPs (dATP, dGTP, dTTP and dCTP), 25 pmol of each primer and 50 pmol oftemplate oligonucleotide resulting in a 114 bp dsDNA MBI-11 fragment.The resulting DNA fragment was cloned into pBCKS-V-S as a BamHI—NsiIfragment basically as described in paragraph (A), resulting in plasmidpBCKS-V-11B7S-F. The only exception was that 2× OPA was used in therestriction enzyme digest reaction.

E. Construction of Plasmid PBCKS-V

Plasmid pBCKS-V was prepared from pBCKS(+). The goal was to eliminateall VspI restriction sites from the original plasmid and use theresulting plasmid for cloning of some of DNA cassettes.

About 1 μg of pBCKS(+) was digested with VspI (Promega) in 50 μlreaction using 1× OPA. The reaction was stopped by phenol/CHCl₃extraction and ethanol precipitation. The resulting DNA was dissolved in50 μl of 1× OPA, 0.2 mM dNTPs and 1 U of Klenow polymerase. The reactionwas incubated at 30° C., for 30 min. and then stopped by phenol/CHCl₃extraction and ethanol precipitation. DNA was then dissolved in 50 μl of1× OPA, 0.5 mM ATP and 15U of T4 DNA ligase and the reaction wasincubated at 10° C. and after 4 hours stopped by incubation at 65° C.for 30 min. Then 20 U of VspI was added to the reaction to digest anyremaining pBCKS(+) molecules and after 3 hours incubation at 37° C., 2μl of the ligation mixture were used to electroporate 40 μl of E. coliMC4100F using a sterile Gene Pulser cuvette (0.2 cm electrode gap) andGene Pulser electroporator apparatus set to 2.5 kV, 200 ohms and 250 μF.After an electroporation pulse, 1 ml of TB media was added to the cellsuspension and bacteria were incubated 1 hour at 37° C. with rigorousshaking. Then 10, 50 and 100 μl of cell suspension were plated onMacKonkey agar plates with 25 μg/ml of Chloramphenicol and incubatedovernight at 37° C. The next day, several colonies were transferred to 2ml of TB and cultivated at 37° C. with vigorous shaking overnight. Thenplasmid DNA was isolated and analyzed by VspI restriction analysis. Allplasmids lacked VspI sites and their size corresponded with thecalculated size of pBCKS-V.

EXAMPLE 6 Construction of Fused Multi-Domain Expression Systems

A. Construction of PET21CBD96-2x11B7

Plasmids pET21CBD96 (0.25 μg) and pBCKS-2x11B7 (2.5 μg) were digestedwith BamHI and HindIII in 1.5× OPA in a 50 μl reaction at 37° C. for 1hour using 2 U of each restriction enzyme and 20U of each enzymerespectively. Both reactions were stopped by phenol/CHCl₃ extraction andethanol precipitation. The resulting DNAs were dissolved in 8 μl ofwater and mixed, then 2 μl of 10 mM ATP, 2 μl of 10× OPA and 2 U of T4DNA ligase were added and the ligation reaction was incubated at 10° C.for 1 hour. Then 2 μl of the ligation mixture was used to electroporate40 μl of E. coli XL1 Blue using a sterile Gene Pulser cuvette (0.2 cmelectrode gap) and Gene Pulser electroporator apparatus set to 2.5 kV,200 ohms and 250 μF. After an electroporation pulse, 1 ml of TB mediawas added to the cell suspension and bacteria were incubated 1 hour at37° C. with rigorous shaking. Then 10, 50 and 100 μl of cell suspensionwere plated on MacKonkey agar plates with 100 μg/ml of Ampicillin andincubated overnight at 37° C. The next day several colonies weretransferred to 2 ml of TB and cultivated at 37° C. with vigorous shakingovernight. Then plasmid DNA was isolated and analyzed, including DNAsequencing by methods known to those skilled in the art. Positive clonepET21CBD96-2x11B7 contained tandem MBI-11 genes fused to cbd96.

B. The use of Serial Cloning Procedure for Construction of FusedMulti-Domain Plasmids

The idea of the serial cloning procedure is that the insertion of theBamHI-MBI-11B7-P-VspI cassette into the BamHI-NdeI sites ofpET21CBD96-2x11B7 and subsequent multi-domain clones always eliminatesthe original NdeI site by NdeI/VspI ligation and a new NdeI site isintroduced with each insertion, which together with BamHI is used forthe next cycle of cloning.

Plasmid pET21CBD96-2x11B7 (0.25 μg) was digested with 2 U of BamHI andNdeI in 2× OPA in 50 μl reaction at 37° C. for 1 hour. PlasmidpBCKS-V-11B7S (2.5 μg) was digested in a 100 μl reaction with 20 U ofBamHI and VspI in 2× OPA at 37° C. for 1 hour. Both reactions werestopped by phenol/CHCl₃ extraction and ethanol precipitation. Theresulting DNAs were dissolved in 8 μl of water and mixed, then 2 μl of10 mM ATP, 2 μl of 10× OPA and 2 U of T4 DNA ligase were added and theligation reaction was incubated at 10° C. for 1 hour. Then 2 μl of theligation mixture were used to electroporate 40 μl of E. coli XL1 Blueusing a sterile Gene Pulser cuvette (0.2 cm electrode gap) and GenePulser electroporator apparatus set to 2.5 kV, 200 ohms and 250 μF.After an electroporation pulse, 1 ml of TB media was added to the cellsuspension and bacteria were incubated 1 hour at 37° C. with rigorousshaking. Then 10, 50 and 100 μl of cell suspension were plated onMacKonkey agar plates with 100 μg/ml of Ampicillin and incubatedovernight at 37° C. The next day, several colonies were transferred to 2ml of TB and cultivated at 37° C. with vigorous shaking overnight. Thenplasmid DNA was isolated and analyzed, including DNA sequencing bymethods known to those skilled in the art. Positive clonepET21CBD96-1s-3x11B7 contained three MBI-11 units with one spacer fusedto cbd96. This was the first cycle of the serial cloning. In the nextcycle pET21CBD96-1s-3x11B7 and pBCKS-V-11B7S were used and cloning wasrepeated resulting in pET21CBD96-2s-4x11B7. Then pET21 CBD96-2s-4x11B7and pBCKS-V-11B7S were used for the next cloning resulting inpET21CBD96-3s-5x11B7 and so on.

In order to accelerate the serial cloning procedure plasmidPBCKS-V-5x11B7S was prepared and each cloning cycle would add five 11B7Sdomains. First the 11B7S insert of pBCKS-V-11B7S was recloned intopTZ18R, resulting in pTZ18R-11B7S. Then this plasmid was used as thedonor of the 11B7S domain for the serial cloning into pBCKS-V-11B7Susing the BamHI-NdeI/VspI strategy. The serial cloning procedure wasrepeated four times resulting in pBCKS-V-5S-5x11B7S. The 5S-5x11B7cassette was then used for construction of CBD96-fused systemscontaining more than fifteen 11B7 domains and also CBD96-fusedmultidomain systems with equal numbers of 11B7 and anionic spacerdomains (Table 2).

The cassette 5S-5x11B7 of pBCKS-V-5S-5x11B7 with anionic spacer domainat the end was cloned into pET21CBD96 using BamHI and KpnI restrictionenzymes resulting in pET21 CBD96-5S-5×11B7. In the second cloning cyclethe same cassette was ligated as BamHI-VspI fragment of pBCKS-V-5x11B7Sinto BamHI-NdeI sites of pET21CBD96-5S-5x11B7 resulting inpET21CBD96-10S-10x11B7. This can be repeated several times to receiveconstructs with 15, 20, 25 etc. 11B7 domains and equal numbers ofanionic spacer domains. Conditions for restriction enzymes, ligation,electroporation and analysis of recombinant plasmids are describedabove.

EXAMPLE 7 Construction of Unfused Multi-Domain Expression Systems

In E. coli, the first amino acid in all proteins is f-methionine.However, this amino acid is not cleaved by CNBr, which means that onepeptide domain released from a multi-domain protein would start withf-methionine. The solution was to create a modified MBI-11 cassetteencoding f-methionine and methionine in tandem at the beginning of thepeptide, so the second one would be cleaved by CNBr. The result was thesynthesis of the special first domain in multi-domain genes, cassetteMBI-11B7F, encoding MTM amino acids at the beginning. This domain wasfused to the spacer domain in pBCKS-V-S resulting in plasmidpBCKS-V-11B7S-F.

Plasmid pBCKS-V-11B7S-F and the relevant pET21 CBD96-multi-domain-11B7plasmids were used for construction of unfused multi-domain MBI-11B7genes. Multi-domain genes were liberated from cbd96 by NdeI-XhoIdigestion and cloned into the VspI-XhoI sites of pBCKS-V-11B7S-Fdownstream of the 11B7S insert. This created a line of unfusedmulti-domain 11B7 genes in plasmid pBCKS-V. These genes were thenrecloned as NdeI-XhoI fragments into pET21a(+) resulting in a series ofpET plasmids capable of expression of multi-domain proteins using the T7promoter system.

Plasmid pBCKS-V-11B7S-F (0.25 μg) was digested with 2 U of NdeI and XhoIin 2× OPA in several 50 μl reactions at 37° C. for 1 hour. Relevantplasmids pET21CBD96-multidomain-11B7 (2.5 μg) were digested in 100 μlreactions with 20 U of NdeI and XhoI in 2× OPA at 37° C. for 1 hour. Allreactions were stopped by phenol/CHCl₃ extraction and ethanolprecipitation. The resultant vector and insert DNAs were dissolved in 8μl of water and mixed, then 2 μl of 10 mM ATP, 2 μl of 10× OPA and 2 Uof T4 DNA ligase were added and ligation reactions were incubated at 10°C. for 1 hour. Then 2 μl of each ligation mixture was used toelectroporate 40 μl of E. coli XL1 Blue using a sterile Gene Pulsercuvette (0.2 cm electrode gap) and Gene Pulser electroporator apparatusset to 2.5 kV, 200 ohms and 250 μF. After an electroporation pulse, 1 mlof TB media was added to the cell suspension and bacteria were incubated1 hour at 37° C. with rigorous shaking. Then 10, 50 and 100 μl of cellsuspension were plated on MacKonkey agar plates with 100 μg/ml ofAmpicillin and incubated overnight at 37° C. The next day severalcolonies were transferred to 2 ml of TB and cultivated at 37° C. withrigorous shaking overnight. Then plasmid DNA was isolated and analyzed,including DNA sequencing by methods known to those skilled in the art.Positive clones contained pET21-multidomain-11B7 plasmids containing 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 21 MBI-11B7 domains.

In the same way, constructs were prepared containing equal numbers of11B7 and anionic spacer domains. By way of illustration:pET21CBD96-5S-5x11B7 was digested with BamHI and XhoI (or HindIII) andfragment 5S-5x11B7 was ligated into BamHI-XhoI (or HindIII) ofpBCKS-V-11B7S-F resulting in pBCKS-V-6S-6x11B7. The BamHI-6S-6x11B7-XhoIcassette of pBCKS-V-6S-6x11B7 was then recloned into BamHI-XhoI ofpET21a(+) resulting in pET21-6S-6x11B7. All cloning procedures and cloneanalysis are described above.

EXAMPLE 8 Construction of Fused Multidomain MBI26 Expression Systems

In our previous work we solved all major problems connected to theconstruction of multidomain cationic peptide expression systems. Thisexample demonstrates we were able to simplify the process, especiallythe need for synthesis of multiple specific DNA cassettes; only onembi26 cassette was prepared and used at the first and last position aswell as for the serial cloning procedure. Plasmids pET21CBD96-1s-26 andpET21CBD96-2s-2x26 were prepared. We tested expression of a combinationof mbi26 and mbi11B7 domains. We performed two cloning cycles, insertingmbi26S cassettes into pET21 CBD96-1S-3x11B7, resulting inpET21CBD96-26S-3x11B7 and pET21CBD96-2x26S-3x11B7. Both constructsexpressed the combined mbi26-11B7 multidomain proteins at good levels.

A. Synthesis of Universal MBI26 Domain

An oligonucleotide CGCC AGGG TTTT CCCA GTCA CGAC GGAT CCGT CTCA TATGACCA TGM ATGG AAAT CTTT CATC AAAA MCT GACC TCTG CTGC TAAA AAAG TTGT TACCACCG CTM ACCG CTGA TCTC TATG CATG CTTA AGCT TCGA TCCT CTAC GCCG GACG C(SEQ ID NO: 26) was synthesized and used as a template for PCR usingprimers CGCC AGGG TTTT CCCA GTCA CGAC (SEQ ID NO:18) and GCGT CCGG CGTAGAGG ATCG (SEQ ID NO:19). PCR was performed in an MJ-Research PTC-100Thermo-cycler in a 50 μl reaction volume with 30 cycles of 94° C., 30sec.; 55° C., 30 sec. and 72° C., 30 sec., 2 U of Taq DNA polymerase,corresponding reaction buffer (10× PCR reaction buffer: 500 mM KCl, 15mM MgCl₂, 100 mM Tris-HCl pH 9), 0.2 mM dNTPs (dATP, dGTP, dTTP anddCTP), 25 pmol of each primer and 50 pmol of template oligonucleotideresulting in a 112 bp dsDNA MBI26 fragment. The resulting DNA fragmentwas cloned into pTZ18R as a BamHI-HindIII fragment as described inExample 2, paragraph (A) resulting in plasmid pTZ18R-26GT. Afterverification of DNA sequence, the BamHI-HindIII mbi26 fragment wasrecloned into pBCKS(+) resulting in pBCKS-26GT.

B. Construction of MBI26 Fused Multidomain System

The first step in construction was a direct fusion of the mbi26 cassetteto cbd96 in pET21CBD96. Plasmids pET21CBD96 (0.25 μg) and pBCKS-26GT(2.5 μg) were digested with BamHI and HindIII in 1.5× OPA (50 μlreaction volume) at 37° C. for 1 hour using 2 U of each restrictionenzyme and 20U of each enzyme respectively. Both reactions were stoppedby phenol/CHCl₃ extraction and ethanol precipitation. Each resulting DNAwas dissolved in 8 μl of water; the two were mixed together with 2 μl of10 mM ATP, 2 μl of 10× OPA and 2 U of T4 DNA ligase and the ligationreaction was incubated at 10° C. for 1 hour. 2 μl of the ligationmixture was used to electroporate 40 μl of E. coliXL1 Blue using asterile Gene Pulser cuvette (0.2 cm electrode gap) and Gene Pulserelectroporator apparatus set to 2.5 kV, 200 ohms and 250 μF. After anelectroporation pulse, 1 ml of TB media was added to the cell suspensionand bacteria were incubated 1 hour at 37° C. with rigorous shaking. Then10, 50 and 100 μl of cell suspension were plated on MacKonkey agarplates with 100 μg/ml of Ampicillin and incubated overnight at 37° C.The next day several colonies were transferred to 2 ml of TB andcultivated at 37° C. with rigorous shaking overnight. Then plasmid DNAwas isolated and analyzed, including DNA sequencing by methods known tothose skilled in the art. Positive clone pET21CBD96-26 contained theMBI-26 gene fused to cbd96.

The second step was preparation of a cassette for the serial cloningprocedure. The mbi26 fragment of pTZ18R-26GT was cloned into pBCKS-V-Sas a BamHI-NsiI fragment basically as described in Example 5 (A),resulting in plasmid pBCKS-V-26S with an mbi26 domain fused to theanionic spacer-encoding sequence. The only exception was that 2× OPA wasused in the restriction enzyme digest reaction. The insert was thencloned into pTZ18R resulting in pTZ18R-26S. This allowed the cloning ofthe BamHI-26S-VspI insert into the BamHI-NdeI sites of pBCKS-V-26S,resulting in PBCKS-V-2S-2x26.

The third step was the actual serial cloning procedure (for details seeExample 6B). Briefly, pBCKS-V-26S was digested with BamHI and VspIresulting in fragment BamHI-26S-VspI, which was ligated into plasmidpET21CBD96-26GT digested with BamHI and NdeI. Positive clonepET21CBD96-1s-2x26 contained two MBI-26 units with one spacer fused tocbd96. This was the first cycle of the serial cloning. In the next cyclepET21CBD96-1s-2x26 and pBCKS-V-26S could be used to preparepET21CBD96-2s-3x26 and so on.

C. Construction and Expression of Combined MBI26-MBI11B7 MultidomainGenes

Plasmid pET21CBD96-1S-3×11B7 was used as a vector for serial cloning ofthe mbi26S domain of pBCKS-V-26S. Briefly, pBCKS-V-26S was digested withBamHI and VspI restriction endonucleases resulting in fragmentBamHI-26S-VspI, which was ligated into plasmid pET21CBD96-1S-3×11B7digested with BamHI and NdeI. Positive clones pET21CBD96-2S-26-3×11B7contained an MBI-26 unit with one spacer fused to three 11B7 units withone spacer. This was the first cycle of the serial cloning. In the nextcycle pET21CBD96-2S-26-3x11B7 and pBCKS-V-26S were used to preparepET21CBD96-3S-2x26-3x11B7.

T7 expression systems were prepared in E. coli MC4100F based on plasmidspET21CBD96-2S-26-3x11B7 or pET21CBD96-3S-2x26-3x11B7 and pGP1-2.Proteins CBD96-2S-26-3x11B7 and CBD96-3S-2x26-3x11B7 were expressed atgood levels after temperature induction.

EXAMPLE 9 Production in Shake Flask Fermentation of Multi-DomainCationic Peptide Fused to Truncated CBD or Unfused Systems

Each of the different pET21CBD96-(n-2)S-nx11B7, pET21CBD96-nS-nx11B7,pET21-(n-2)S-nx11B7 and pET21-nS-nx11B7F constructs (where n=number ofcopies, S represents the anionic spacer, and 11B7 or 11B7F representsthe cationic MBI-11B7 peptide) were expressed in E. coli strain MC4100F.

All fermentations are done in TB broth, which is prepared as follows: 12g of Trypticase Peptone (BBL), 24 g of yeast Extract (BBL) and 4 ml ofglycerol (Fisher) is added to 900 ml of Milli-Q water. The material isallowed to dissolve and 100 ml of 0.17 M KH₂PO₄ (BDH), 0.72 M K₂HPO₄(Fisher) is added. The broth is autoclaved at 121° C. for 20 minutes.The resulting pH is 7.4.

A one liter Erlenmeyer flask with 170 ml medium, containing 100 μg/mlampicillin (Sigma-Aldrich Corp.) and 30 μg/ml kanamycin A (Sigma-AldrichCorp.), was inoculated with the relevant 0.5 ml frozen stock and shakenat 300 rpm in a shaking incubator (model 4628, Lab Line InstrumentInc.), at 30° C. for 16 hr.

The culture was then transferred to a 2.0 L flask with 330 ml fresh TBmedium (no antibiotics), preincubated at 30° C. After dilution, proteinexpression was induced by raising the culture temperature to 42° C. andshaking at 300 rpm for another 5 to 7 hours. The pH was kept between 6.7and 7.1 using 30% ammonium hydroxide. Bacteria were fed at least twiceduring induction with 0.5 g glucose per flask. Cells were harvested bycentrifugation (Sorvall® RC-5B) at 15,000×g for 15 minutes and cellpellets were stored at −70° C. prior to cell lysis.

EXAMPLE 10 Crude Fractionation AND Inclusion Bodies Isolation

The bacteria produce the multi-domain proteins as insoluble inclusionbodies. To release and isolate the inclusion bodies, the harvested cellswere suspended in 200 ml buffer (50 mM Tris-HCl, 10 mM EDTA, pH 8.0) andlysed by sonication (Vibra-Cell™, Sonic and material Inc.) five timesfor 45 seconds, on ice, then centrifuged (Sorvall® RC-5B) at 21,875×gfor 15 min at 4° C. The pellet was homogenized (PolyScience, Niles, Ill.USA) in 160 ml of lysis buffer (20 mM Tris-HCl, 100 μg/ml lysozyme, pH8.0) and incubated at room temperature for 45 min. Next Triton X-100 wasadded (1% v/v), and the mixture was homogenized thoroughly andcentrifuged at 21,875×g for 15 min at 4° C. The inclusion bodies pelletwas resuspended in 200 ml of 0.1 M NaCl, homogenized, and precipitatedby centrifugation as described above, then resuspended in 200 ml waterand precipitated again by centrifugation. At this stage, the inclusionbodies contained greater than 70% fusion protein.

EXAMPLE 11 Releasing of Cationic Peptide by Chemical Cleavage

The isolated inclusion bodies were dissolved in 70% formic acid (100 mgwet weight IB per ml), then CNBr was added to a final concentration of0.1 to 0.15 M. The cleavage reaction which allowed the release ofcationic peptide from the fusion protein and spacer was performed undernitrogen and with stirring, in the dark, for 4 hr. Next the reactionmixture was diluted with 15 volumes of Milli-Q water and dried in arotovap machine (Rotovapore, R-124VP, BUCHI Switzerland). The driedpellet was then dissolved in 10 ml of 7-8 M urea and insoluble materialswere separated by centrifugation at 21,875×g for 15 min.

At this stage, the soluble materials, at acidic pH (2-3.3) and lowconductivity (1-5 mS), contain the homoserine lactone form of thecationic peptide. This material was further purified using achromatography procedure.

EXAMPLE 12 Free Cationic Peptide Purification

The purification of the homoserine lactone form of MBI-11B7 peptide wasperformed on a BioSys™ 2000 chromatography work station (BeckmanInstruments, Inc.), using Fast Flow Q-Sepharose anion exchange resin(Pharmacia Biotech AB) packed in an XK column (1.6×11 cm). The columnwas equilibrated with 2 column volumes (CV) of 1 M NaOH at a flow rateof 9 ml/min, followed by a water wash. Conductivity, pH and absorbencyat 280 nm were monitored. When the conductivity dropped below 5 mS, thedried cleavage materials, dissolved in 7-8 M urea, were loaded onto thecolumn and washed with 4 M urea. The unbound pure cationic peptideflowed through the column and was monitored as the leading peak. Whenthe absorbance dropped to baseline, the bound material (i.e.,impurities) was washed off the column with 1 M NaOH and appeared as thesecond peak (FIG. 9).

The flow-through peak was collected and pooled and the pH was adjustedto 7.0-7.5 with 0.2 N HCl. The sample was analyzed for purity by reversephase HPLC (FIG. 11), using a C8 column (4.6×10, Nova-Pak, Waters) andby acid-urea gel electrophoresis (West and Bonner, Biochemistry 19:3238,1980). The identity of the MBI-11B7 peptide was confirmed by massspectrometry to show that the flow through peak represents thehomoserine form of the MBI-11B7 peptide.

EXAMPLE 13 Urea Separation and Further Purification

The separation of the urea from the purified peptide utilized ahigh-throughput reverse phase chromatography technique by using theBioCAD™ (PerSeptive Biosystems Inc.) perfusion chromatographyworkstation and Poros® R-II 20 column, 4.6×100 mm (PerSeptive BiosystemsInc.). About 10 mg of the peptide were applied on the column at 5ml/min, followed by equilibration of the column with 0.1% TFA. Thepeptide was eluted from the column by a gradient of increasingacetonitrile from 0 to 50% for 10 minutes at a flow rate of 5 ml/min.The peak of the further purified and urea free peptide was collected andlyophilized.

EXAMPLE 14 Bactericidal Activity of MBI-11B7CN Peptide and itsHomoserine/Homoserine Lactone Isoforms

A comparison of anti-microbial activity between chemically andrecombinantly synthesized cationic peptide was carried out.

The antimicrobial activities of the chemically synthesized MBI-11B7CNpeptide and recombinant DNA synthesized MBI-11B7HSL (homoserine lactoneform) and MBI-11B7HS (homoserine form) peptides were tested againstvarious gram-negative and positive strains of bacteria, includingantibiotic resistant strains. The Agarose Dilution Assay was performedas described in “Methods for Dilution Antimicrobial Susceptibility Testsfor Bacteria That Grow Aerobically-Fourth Edition; Approved Standard”NCCLS document M7-A4 (ISBN 1-56238-309-4) Vol. 17, No 2 (1977).

The agarose dilution assay measures antimicrobial activity of peptidesand peptide analogues, which is expressed as the minimum inhibitoryconcentration (MIC) of the peptides.

In order to mimic in vivo conditions, calcium and magnesium supplementedMueller Hinton broth is used in combination with a low EEO agarose asthe bacterial growth medium. Agarose, rather than agar, is used as thecharged groups in agar prevent peptide diffusion through the media. Themedium is autoclaved and then cooled to 50° C.-55° C. in a water bathbefore aseptic addition of anti-microbial solutions. The same volume ofdifferent concentrations of peptide solution are added to the cooledmolten agarose, which is then poured to a depth of 3-4 mm.

The bacterial inoculum is adjusted to a 0.5 McFarland turbidity standard(PML Microbiological) and then diluted 1:10 before application on to theagarose plate. The final inoculum applied to the agarose isapproximately 10⁴ CFU in a 5-8 mm diameter spot. The agarose plates areincubated at 35° C.-37° C. for 16 to 20 hours.

The MIC is recorded as the lowest concentration of peptide thatcompletely inhibits growth of the organism as determined by visualinspection. Representative MICs for the cationic peptides againstvarious bacterial strains are shown in Table 3. TABLE 3 MINIMUMINHIBITORY CONCENTRATION (MIC) VALUES FOR MBI-11B7CN (CARBOXY-AMIDATED),MBI-11B7HSL (HOMOSERINE LACTONE FORM) AND MBI-11B7HS (HOMOSERINE FORM)PEPTIDES, AGAINST VARIOUS GRAM-NEGATIVE AND GRAM-POSITIVE BACTERIASTRAINS MIC (μg/ml) Organ- 11B7CN 11B7HSL 11B7HS Organism ism # Source92A1 203B1 203B1 A. calcoa- AC2 ATCC 2 4 2 ceticus E. cloacae ECL7ATCC >64 >64 >64 E. coli ECO5 ATCC 8 8 32 K. pneumoniae KP1 ATCC 8 8 32P. aeruginosa PA4 ATCC >64 >64 >64 S. malto- SMA2 ATCC 32 32 64 philiaS. marcescens SMS3 ATCC >64 >64 >64 E. faecalis EFS1 ATCC 2 1 2 E.faecalis EFS8 ATCC 16 16 32 S. aureus SA14 Bayer 4 1 2 S. aureus SA93Bayer 1 1 1 S. epider- SE10 Chow 2 4 8 midis

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention, which isdefined by the following claims.

1. A fusion protein expression cassette, comprising a promoter operablylinked to a nucleic acid molecule that encodes a fusion proteincomprising a structure of (cationic peptide)-[(cleavage site)-(cationicpeptide)]_(n) with n being an integer having a value between one andthree, wherein the cationic peptides have antimicrobial activity and thecleavage site can be cleaved by low pH or by a reagent selected from thegroup consisting of2-(2-nitrophenylsulphenyl)-3-methyl-3′-bromoindolenine, hydroxylamine,o-iodosobenzoic acid, Factor Xa, thrombin, enterokinase, collagenase,Staphylococcus aureus V8 protease, endoproteinase Arg-C, and trypsin. 2.The expression cassette of Claim 1 wherein said fusion protein comprises3 cationic peptides.
 3. The expression cassette of claim 1 wherein saidfusion protein comprises 4 cationic peptides.
 4. The expression cassetteaccording to claim 1 wherein the fusion protein is cleaved byendoproteinase Lys-C.
 5. The expression cassette according to claim 1wherein the cationic peptide has up to 35 amino acids comprising thesequence of I L K K W P W W P W R R K (SEQ ID NO: 35) or 1 L R W P W W PW R R K (SEQ ID NO:36).
 6. The expression cassette according to claim 1wherein the cationic peptide is I L R W P W W P W R R K (SEQ ID NO:36).7. The expression cassette according to claim 1 wherein said promoter isselected from the group consisting of lacP promoter, tacP promoter, trcppromoter, srpP promoter, SP6 promoter, T7 promoter, araP promoter, trpPpromoter, and λ promoter.
 8. The expression cassette according to claim1 wherein said nucleic acid molecule also encodes a carrier protein. 9.The expression cassette according to claim 8 wherein the carrier proteinis selected from cellulose binding domain, glutathione-S-transferase,outer membrane protein F, β-galactosidase, protein A, or IgG-bindingdomain.
 10. The expression cassette according to claim 8 wherein saidcarrier protein is less than 100 amino acid residues in length.
 11. Theexpression cassette according to claim 1 wherein said nucleic acidmolecule also encodes an anionic spacer peptide component comprising astructure of (cationic peptide)-[(cleavage site)-(anionic spacerpeptide)-(cleavage site)-(cationic peptide)]_(n).
 12. The expressioncassette according to claim 11 wherein said anionic spacer lacks acysteine residue.
 13. A recombinant host cell comprising the expressioncassette according to claim
 1. 14. The recombinant host cell of claim 13wherein the expression cassette is contained in an expression vector.15. The recombinant host cell of claim 13 wherein said host cell is ayeast, fungi, bacterial or plant cell.
 16. The recombinant host cell ofclaim 15 wherein said bacterial host cell is Escherichia coli.
 17. Amethod of producing a fusion protein, comprising culturing therecombinant host cell of claim 13 under conditions and for a timesufficient to produce the fusion protein.
 18. The method according toclaim 17 wherein the fusion protein is cleaved at the cleavage sites torelease the cationic peptides.
 19. The method according to claim 17wherein the cationic peptide is I L R W P W W P W R R K (SEQ ID NO:36).